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The Dune Encyclopedia
This article or section refers to elements that appear exclusively in The Dune Encyclopedia.
Staivan was a theoretical wave mechanic and chief engineer of House Varrik. He lived around 3832 BG.
That year, Ibrahim Vaughn Holtzman made his third return near Liesco II which by that time, was a prison planet. Holtzman was detected and mistaken for a communication device constructed by the prisoners and in order to protect himself, he transmitted his discoveries of the Planar Effect.
Staivan was contacted by the Provincial Warden on Maktiun III, in order to investigate the recordings. Staivan found nothing inconsistent in the theories ane mechanisms of the tapes and tried to follow the instructions, generating thus a Node for this "Planar Effect".
Thus he the first manufactured Shield, which was a simple flat effect, about a meter in diameter, admitting radiation in all but the long radio frequencies. Its existence was not detectable until Staivan accidentally dropped a screwdriver which lowered gently through the plane of the effect. Staivan then reported that these mysterious messages were no hoax.
He then took the apparatus into the courtyard to test its resistance to a variety of missiles of increasing power, including a workman's cutting laser, and learned that the Planar Effect did not mix well with coherent light.
Holtzman was eventually contacted, and the Planar Effect spread like wildfire. Eventually the Planar Effect led to the creation of the Defensive Shield.
- Dune Encyclopedia, entry Ibrahim Vaughn Holtzman

subset: commoncrawl


text:
VokAgent, the Vocables Agent - A language neutral vocables coach for each pupil
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text:
Mick Jagger to perform live for the first time at the Grammys
Mick Jagger, lead singer of legendary rock band The Rolling Stones, will score a first in musical performance as part of the 53rd Grammy Awards, on February 13 in Los Angeles.
Jagger will take the stage as part of a tribute to recently deceased soul legend Solomon Burke, who died of natural causes Oct. 10 at the age of 70.
In 1986, Mick Jagger and his group made a brief appearance via satellite at the 28th Grammy Awards, from London, when Eric Clapton presented their award for lifetime achievement throughout their careers.
Recording Academy president and CEO Neil Portnow said he's ecstatic about Jagger's appearance.
"We're thrilled, delighted, excited and very much looking forward to Mick doing his debut on the Grammy stage," Portnow told The Associated Press. "It's extraordinary to many of us that the fact is, this will be the first time; he has never performed on the Grammy stage."
This will be the first performance at the Grammy Awards for Mick Jagger and many other artists, including Arcade Fire, Justin Bieber, Jaden Smith, the rapper BoB, Miranda Lambert, Bruno Mars, Gwyneth Paltrow, Janelle Monáe and the British band Muse .
Lady Gaga, Katy Perry, Eminem, Cee Lo Green and Lady Antebellum will also perform at the gala which will be shown live on CBS, February 13, from 20 pm
Eminem is the artist who has garnered the highest number of nominations for the 53rd edition. He was cited 10 times with the success of his latest album, Recovery.


text:
Happy shopping!!
Monday, November 26, 2012
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It's that time of year again - sale time! To celebrate the holiday season, I'm having a Cyber Monday sale in my Etsy store. All prints are 20% off, so hop on over and take a look around. But hurry - for a limited time only!
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text:
What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
How can I hide empty fields on the edited email template?Asked by mgcaledonian13 on December 21, 2014 at 06:59 AM
I want to do the same but as you point out if this Template is edited at all it then upsets the hide empty emails. Does mean even if the "Question & Answer" is removed.
I would think that a lot of people would be glad of the ability to remove some of the text that means nothing to the user in a Response without making the Template "dirty". Would this feature be considered at all? Is there way to work around this?
JotForm Support
Hi Willy,
Everything outside of the data table can be edited. Unfortunately, the Question and Answer headings fall within the said table, as can be seen below:
Also see this thread and our guide on the feature.
I could submit a ticket requesting that the aforementioned headings be excluded from the data table but are there any other elements you would like to be excluded?
Well that is good news about data outside the table. You asked if there was anything else, there is, can the hidden fields be left out, they are there just for reference purposes. For instance an order number that would be incremental and only needed by the recipient of the notification but not the user.
Willy Scott
Unfortunately for now it seems not possible to hide empty fields and still edit the email so that hidden fields are not displayed.
I forwarded a feature request to our developers so they can consider its future implementation.
You'll be contacted via this thread once they have an update on this.
Let us know if you need more assistance.
Any update to this??? Seems silly to have to use the default notification template to achieve hiding the empty fields.
JotForm Support
When the 'Hide Empty Fields on Emails' option is checked you cannot edit the Questions/Answers table, but everything else is editable.
This is not likely that we will re-implement mentioned behavior in the near future. Thank you.


text:
Sitter in Marietta
Are you looking for reliable, safe and convenient Childcare? My name is Cherry, I am a mother of a 4 year old daughter and have many years in Childcare experience. I offer drop in hours as well as permanent Childcare. Only ages newborn-5 years old. Limited available space for newborns. Hours are as followed.. Sunday- Thursday 5pm-6am Prices are as followed.. Drop in $35.00 up to 10 hours $5 per hour after the 10th hour Weekly $85.00 for five days 10 hours each day with parents providing their own meals with meals is $95 a week If more then one child we can discuss prices. Payments are due at the time of drop off or they cannot stay, sorry. For more info please feel free to call show contact info
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Light Meal Preparation
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|Jim Jagielski <...@jaguNET.com>
|[ANNOUNCE] Apache 1.3.17 Released!
|Tue, 30 Jan 2001 02:15:23 GMT
Apache 1.3.17 Released The Apache Software Foundation and The Apache Server Project are pleased to announce the release of version 1.3.17 of the Apache HTTP server. Version 1.3.15 was not released due to problems when tagging the tree. Version 1.3.16 was not released due to some last minute bug fixes required after it was tagged. This version of Apache is primarily a bug fix release, addressing some broken functionality present in the 1.3.14 release and various Win32 issues. There are, however, a few new features and improvements. A summary of the new features is given at the end of this document. We consider Apache 1.3.17 to be the best version of Apache available and we strongly recommend that users of older versions, especially of the 1.1.x and 1.2.x family, upgrade as soon as possible. No further releases will be made in the 1.2.x family. Apache 1.3.17 is available for download from http://httpd.apache.org/dist/ Please see the CHANGES_1.3 file in the same directory for a full list of changes. Binary distributions are available from http://httpd.apache.org/dist/binaries/ As of Apache 1.3.12 binary distributions contain all standard Apache modules as shared objects (if supported by the platform) and include full source code. Installation is easily done by executing the included install script. See the README.bindist and INSTALL.bindist files for a complete explanation. Please note that the binary distributions are only provided for your convenience and current distributions for specific platforms are not always available. The source and binary distributions are also available via any of the mirrors listed at http://www.apache.org/mirrors/ For an overview of new features in 1.3 please see http://httpd.apache.org/docs/new_features_1_3.html In general, Apache 1.3 offers several substantial improvements over version 1.2, including better performance, reliability and a wider range of supported platforms, including Windows 95/98 and NT (which fall under the "Win32" label). Apache is the most popular web server in the known universe; over half of the servers on the Internet are running Apache or one of its variants. IMPORTANT NOTE FOR WIN32 USERS: Over the years, many users have come to trust Apache as a secure and stable server. It must be realized that the current Win32 code has not yet reached the levels of the Unix version, but is of acceptable quality. Any Win32 stability or security problems do not impact, in any way, Apache on other platforms. Apache 1.3.17 Major changes The bug fixes are: * Correct handling of request byte ranges as specified by RFC 2616. * Restore functionality broken by the mod_rewrite security fix: rewrite map lookup keys and default values are now expanded so that the lookup can depend on the requested URI etc. * PHP now works under Unixware 7. * Eliminate caching problems of mod_autoindex results. * Win32 CGI execution on all Windows platforms is corrected. * NetWare file and path fixes. The main new features include: * Add a new LogFormat directive, %c. * mod_status now respects ?refresh=n of 1 or greater. * mod_autoindex fixes browser caching by sending LastModified and ETag headers if IndexOptions TrackModified is enabled. Selected new features that relate to Windows platforms: * Complete handling of ScriptInterpreterSource Registry execution. * NT and 2000 services can be started with -D FOO style options. * The close console window button/menu option on 95 and 98 works. Selected new features relating to other platforms: * Better discovery of dbm_open() under Linux. * Linux 2.2.x and later do not need a serialised single listener. -- =========================================================================== Jim Jagielski [|] email@example.com [|] http://www.jaguNET.com/ "Casanova will have many weapons; To beat him you will have to have more than forks and flatulence."
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text:
A proton is a positively charged subatomic particle found in the nucleus of an atom.
What it doesEdit
First of all, the number of protons in the nucleus decides what the element is. For example, if there are 3 protons, then it is lithium (Li), if there are NOT 3 protons, then it is NOT lithium. This is the atomic number, it is NOT to be confused with atomic mass, which is mass of protons AND neutrons in the nucleus combined.
If the protons are positive, then why doesn't the nucleus explode from the rule "like charges repel"?Edit
Good fucking question! Your answer: The neutrons are neutral in charge! Their job is to keep the protons together like velcro keeps 2 likecharge magnets together, it only works at a very small proximity, it is called "strong nuclear force"!
How it was discoveredEdit
Geiger-Marsden Experiment was an experiment thought up by Ernest Rutherford, and carried out by Hans Geiger and Marsden! It involved trying to test the plum pudding model (which taught that electrons were dotted around in a pudding of positive charge) by blowing up one of the "pudding" atoms of a gold leaf with alpha particles.
What ACTUALLY happened was different from what was expected, the rays of alpha particles were being reflected and some of them went through the gold leaf!
This proved that the positive alpha particles were being repelled by a positive core, but were being let through partially because much of the atom was unoccupied by matter.


text:
- About
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Data from: Chronic malnutrition favours smaller critical size for metamorphosis initiation in Drosophila melanogaster
Date Published: November 10, 2011
DOI: http://dx.doi.org/10.5061/dryad.k4c8dk31
Content in the Dryad Digital Repository is offered "as is." By downloading files, you agree to the Dryad Terms of Service. To the extent possible under law, the authors have waived all copyright and related or neighboring rights to this data.
|critical weight data
|21 times
|This data was collected in our laboratory using populations of D. melanogaster experimentaly evolved for increased adaptation to chronic larval malnutrition. The data file includes the larval wet weight and proportion pupariating at 7 time points for 12 populations (6 selected-S1 to S6;and 6 control C1 to C6).
|critical weight data.xls (31.23 Kb)
|View File Details
When using this data, please cite the original publication:
Additionally, please cite the Dryad data package:
We encourage organizations to:


text:
10036131, Panel
Please help our community — upload your photos of 10036131.
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I would like to know the following:
If I have 1 NIC and 2 internet connections (and thus 2 different IPs on the same physical line), how can I order a certain application to use a certain IP (windows XP pro sp2). Is there any software that would let me chose which connection to assign to say MSN or something?
Best regards and thank you for your anwsers.
1 Reply
Feb 29, 2008 at 1:36 UTC
You mention that you have 2 Internet connections; what sort of device does each connection terminate to; the Windows box itself, or some sort of firewall device(s)?
In other words, does your Windows machine have 1 NIC with 2 different (public) IP addresses configured? If so, you could always add static routes *based on the destination* (this isn't really routing different protocols along different paths, but it's as close as you may be able to get).
Windows XP has the QoS (Quality of Service) packet scheduler (http:/
This discussion has been inactive for over a year.
You may get a better answer to your question by starting a new discussion.


text:
|Publication number
|Publication type
|Application number
|Publication date
|Mar 1, 1983
|Filing date
|May 20, 1981
|Priority date
|May 20, 1981
|Fee status
|Publication number
|Nicholas Catsimpoolas
|Original Assignee
|Nicholas Catsimpoolas
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (6), Referenced by (78), Classifications (11), Legal Events (7)
|External Links: USPTO, USPTO Assignment, Espacenet
In U.S. Pat. No. 4,124,470 there is described an automatic apparatus for zone or density gradient electrophoresis. The apparatus utilizes a turntable onto which are loaded empty electrophoresis tubes which are filled in serial fashion with sample and electrophoresis medium utilizing an automatic diluter. Each of the tubes has conductive means at both ends and when voltage is supplied an electrophoretic pattern is produced in the tubes. The turntable transports the tubes from the loading station to a scanning station while the electrophoresis separation is proceeding. The electrophoretic patterns are scanned by means of an optical probe which is dipped into the electrophoresis tube without disturbing the bands. Other types of protein (isoenzymes) are measured after dispensing of a selective reagent.
A tube gel electrophoresis device is described in U.S. Pat. No. 4,048,049 which comprises a number of concentrically mounted gel tubes. The tubes extend between an upper buffer chamber and a lower buffer chamber. The lower buffer chamber is operatively connected to a cooling core so as to provide heat transfer during the electrophoresis. An advantage ascribed to this apparatus is that it may be assembled or disassembled without draining the buffer and coolant. Morever the individual tubes may be removed from the assembly during operation without similar draining of the buffer and cooling chambers. A variety of gel tube sizes can be accommodated by using interchangeable upper buffer chambers. The tubes do not rotate during operation but remain fixed in their relative positions. A similar multiple tube gel electrophoresis apparatus for handling a number of samples simultaneously is disclosed in U.S. Pat. No. 3,867,271.
In U.S. Pat. No. Re. 27,552 an apparatus for density gradient electrophoresis is described. An important feature of this apparatus is the use of electrodes immersed in electrolytes disposed near the top and the bottom of the vertical gradient tube. Semi-permeable membranes are utilized to permit ionic current flow from the electrolyte solutions to the fluid columns contained within the vertical tube while isolating such solutions from the columns.
U.S. Pat. No. 4,199,428 provides a sample feeder which can be used for sequentially delivering multiple samples to a sample applicator of an electrophoresis system.
An automatic multiple-sample applicator for a two-dimensional gel electrophoresis system is described in U.S. Pat. No. 4,061,561. The multiple-sample applicator coacts with a tank cover allowing an operator either to depress a single button thus causing multiple samples to be simultaneously deposited on the gel or to depress one or more sample applicators separately by means of a separate button for each applicator. The sample applicator picks up multiple specimens from corresponding wells in the sample holder using capillary action to fill when contact is made by the applicator with the fluid surface. The electrophoretic gel is held in a tray and the samples are transferred to the gel through the applicator tips. These tips can cut into the gel and thus they eliminate the need for prepared slot formation on the gel.
U.S. Pat. No. 4,130,471 provides a microelectrophoretic apparatus and process which employs a multi-sample applicator capable of applying 10 samples consecutively or simultaneously and is adapted to be used with the apparatus of U.S. Pat. No. 4,061,561 discussed above for two dimensional gel electrophoresis.
Alternative systems used in the art to provide continuous electrophoretic separations in an automated mode utilized moving belts or film covered with the electrophoretic medium and sample disposed on one surface and passing the belt or film through the electrophoretic chamber. Representative systems of this type of arrangement are found in U.S. Pat. No. 3,133,009 (coated tape); U.S. Pat. No. 3,896,021 (cellulose acetate coated tape); U.S. Pat. No. 4,059,501 (coated moving belt) and U.S. Pat. No. 4,198,284 (pair of endless belts carrying sample on film).
The objective of the electrophoretic system of the present invention is to provide a convenient, rapid and reproducible means for performing high-resolution, preparative or analytical electrophoresis in an automated manner and with second dimension capability. The total system consists of a number of sub-systems or sub-units combined into an automated microprocessor controlled system. The objective of ease of operation is accomplished by:
(1) providing automatic media preparation for the first dimensional electrophoresis;
(2) utilizing multiple, automatic sample application for both dimensions;
(3) automatically controlling parameters of operation such as cooling, fluid flow, movement of fractions, cleaning, and sterilization solutions, etc.
The objective of speed is met by use of a multiple column system which processes up to twelve samples per complete revolution. In addition automatic media preparation (1st dimension) and the availability of pre-prepared media as articles of commerce greatly reduces the time for the overall operation.
The object of reproducibility is accomplished by employing fully automated conditions and sequence controls of electrophoresis and the standardized format and grid locator used for precise identification and quantitation.
The first dimensional system utilizes isoelectric focusing or electrophoresis, or isotachophoresis using density gradients in glass columns. These columns are supported in circular configuration within a rotating concentric turntable or carousel. Means are provided to allow needed fluid flow of buffers and cooling solution throughout the rotational cycle during which electrophoresis of the sample takes place. Distinct stations are provided at selected points in the carousel to effectuate one of the following functions:
Deposition of gradient density solution in column
Deposition of sample/deposition of buffers
Gradient/fraction evacuation and monitoring
Washing, rinsing and drying columns.
In a preferred embodiment the assemblies and components of the first dimensional system include the following:
1. Gradient density maker;
2. Sample applicator;
3. Buffer applicator;
4. Carousel assembly and motor;
5. Cooling assembly;
6. Cleaning apparatus assembly;
7. Power supply assembly;
8. Evacuation monitor assemblies;
9. Fraction collector assembly;
10. Recorder assembly;
11. Microprocessor control assembly.
The fraction collector assembly is of a novel design and construction which allows up to about 50 discrete fractions to be retained in a single holder. The holder comprises up to 50 individual sample chambers which are each in fluid flow connection with a corresponding individual capillary sample applicator. When an air tight cover is inserted over the holder and pressure applied, the individual fraction samples are applied directly to a second dimension electrophoresis gel in preselected and uniformly distributed positions. In cases where the fractions provided to the fraction collector are monitored by UV or pH, and thus the various volumes in the individual sample chambers are non uniform, a novel multi-element siphoning system is provided to equalize the volumes so as to allow uniform sample quantities to be used in the second dimension electrophoresis.
The fraction collector holder is designed and constructed so as to be attachable to the tank which holds the electrophoretic gel slabs or the slabs themselves for the purpose of properly distributing the fractions to the gel in their appropriate places. Preferably the tank will accommodate the same number of slabs as the number of gradient columns in the carousel of the first dimensional system. Thus all fractions which are collected from a run on the first dimensional system can be transferred in a single operation and run simultaneously in the second dimensional system. Up to six hundred separate fraction samples can be run in this manner during one cycle of the total system. The fraction collector holder is removed after sample application and prior to initiation of the second dimension electrophoresis.
Basic elements which comprise the second dimensional system include the following:
electrophoresis media
sample applicator
cooling system
visualization process
power supply
Additional objects and features of the invention will appear from the following description in which the preferred embodiment has been set forth in detail in conjunction with the accompanying drawings.
FIG. 1 is a schematic block diagram representation of the first dimensional system showing control interactions between the main subcomponents;
FIG. 2 is an isometric view of the upper collector assembly of the first dimensional system carousel which provides continuity of flow during operational rotation.
FIG. 3a is a view of the engaging surface of a stationary disk or surface which engages the rotating portion of the carousel and by means of positioned groove and hole openings serves to control fluid flow during rotation.
FIG. 3b is a view of the engaging surface of a rotating disk or surface which engages the stationary disk or surface and contains a series of concentric holes which provide fluid flow connections for the various functions associated with the electrophoresis processes.
FIG. 3c is a cross-sectional view of the lower collector assembly showing the fluid inlets and outlets for the carousel and the assembled stationary and rotating disks.
FIG. 3d is a cross-sectional detail view of the stationary and rotating disks showing the Teflon ring washer used to provide isolation between adjacent rings of holes in each disk.
FIG. 4 is a side isometric view of the carousel assembly showing the relationship of the electrophorectic tubes, the fluid connections and the support means.
FIG. 5 is a top and cross-sectional view of ring section assembly of the upper section of the carousel showing a single column construction comprising the outer cooling column, the inner gradient electrophoresis column and the electrode assembly and the fluid inlets.
FIG. 6 is a top and cross-sectional view of the ring section assembly of the lower section of the carousel showing a single column construction comprising the outer cooling column, the inner gradient electrophoresis column and the electrode assembly and the fluid inlets for each.
FIG. 7 is a schematic view of one embodiment of a density gradient generator for the first dimensional electrophoresis.
FIG. 8 is a schematic view of one embodiment of a sample applicator assembly for the first dimensional electrophoresis.
FIG. 9 is a schematic view of one embodiment of the fraction collection assembly of the first dimensional electrophoresis.
FIG. 10 is a front view of the fraction collector sample holder with pressure manifold cover.
FIG. 11 is a front view of the sample volume equalizer with vacuum manifold.
FIG. 12 is a side view of the sample volume equalizer.
FIG. 13 is a diagrammatic view of the second dimensional system showing the multiple fraction collector cassettes filling atop the second dimensional gels supported within the electrophoresis tank.
The preferred embodiment of the present invention is more readily understood by reference to the aforesaid Drawings.
In FIG. 1 there is shown a block diagram depicting the main assembly components of the first dimension electrophoresis system and the control elements providing manual or automatic operation. The microprocessor will provide a complete sequence of events needed to complete the first dimension electrophoresis processes. The microprocessor monitors and controls all functions to be performed and designates the time sequence for each event. In addition to sequence controls monitored by the aforesaid microprocessor, the system is provided with manual controls which can override the microprocessor with respect to certain steps in the process. Such manual controls include:
Photometer control
Carousel controls including controls for manually advancing the carousel to a specific, desired location, a home control which relocates the carousel to provide column #1 to station #1, which is the gradient density solution disposition station, and control for forward and reverse direction of the carousel
Collector controls for manual X-Y positioning of the fraction collector
Carousel/Collector Column/Data Display
Electrophoresis Time/Sequence Controls for individual setting of the time for electrophoresis
Column Selection/Monitor Controls
Power Supply Controls to control voltage level at electrodes.
Additional controls not shown but which can be employed, if desired, include controls for cleaning the carousel, reservoirs, and monitoring/collecting tubing and controls for controlling flow of the cooling system.
As seen in FIG. 1 rotation of the carousel assembly is carried out by activation of the motor control by signals provided either by the microprocessor under automatic control or from the manual carousel controls discussed above. A stepping motor is utilized in the preferred embodiment to effect the rotation from station to station. When a twelve column carousel is used then an equivalent number of stations are provided and the stepping motor provides a 30� rotation per step.
The initial station in the first dimension electrophoresis system is the gradient disposition station. When an empty, cleaned electrophoresis inner column on the carousel is positioned at this station and the gradient making cycle is initiated under microprocessor control the gradient deposition station applicator is put in the activated or down position to provide a fluid flow conduit between the gradient density maker, and the carousel inner column at station #1. The gradient density maker mixes solutions such as sucrose of Ficoll of selected densities (i.e. 5% to 40%) to generate a solution that has a density which varies linearly between the aforesaid limits at a predetermined rate. The solution is then deposited in the inner column of the carousel assembly. During the gradient density application, the carousel assembly is stationary until the gradient is completely deposited and the applicator is lifted up to its stand-by position. Particular details about the construction and operation of the gradient deposition station are provided later in the specification.
The second station in the carousel rotation cycle is the sample desposition station. In a preferred embodiment the sample is withdrawn from a conventional individual sample holder cuvette within a sample collector by using a device employing X-Y positioning. The sample is then deposited in the inner tubes in the carousel after the particular column was filled with the gradient density and shifted to the next station for sample application. As was the case with the gradient density applicator, the application of the sample is again done by means of an up/down control that positions the applicator nozzle into the inner tube, just above the previously deposited gradient solution.
To avoid mixing of samples between applications, the sample applicator should be cleaned completely before the next sample application to avoid contamination and mixing. Further details of the construction and operation of the sample applicator station are provided below.
Activation of the stepping motor and rotation of the carousel to the next station which provides a flow of buffer solution to the upper chambers and lower chambers of the carousel. The buffer solutions are stored in two reservoirs, one for each of the aforesaid group of chambers. In the carousel, the buffer solution provides ionic contact between the electrode assemblies at each of the columns and the semipermeable membranes which provide isolation between the sample containing gradient solution and the buffer. Both lower and upper buffer chambers in each column have separate inlet/outlet ports that allow filling and emptying the buffers as well as recirculating the buffer solutions during the electrophoresis process.
The electrophoresis is initiated when the carousel is rotated to the next (fourth) station. A high voltage potential is applied across the electrodes under control of the power supply control. This potential is maintained during the entire electrophoresis procedure even during column rotation from station four to station ten.
When the subject column is rotated to station eleven the electrophoresis has terminated and under control of the collector controls the contents of the electrophoresis tube is evacuated through the lower collector and fractions taken by the fraction collector assembly. Fraction cut-off can be pre-set on a volume basis, i.e., fractions are taken after a predetermined number of milliliters are evacuated or alternatively the electrophoresis medium can be monitored by one or more desired parameters such as ultraviolet absorption or pH and fraction cuts made when these parameters change by predetermined amount. The fraction collection assembly is capable of obtaining up to 50 discrete fractions from each individual column. Thus during one complete carousel cycle a total of 600 fractions can be collected.
The fraction collector assembly utilizes X-Y positioning and up/down sequence controls to provide deposition of fractions to a designated fraction collector cuvette, most preferably in a uniquely designed cuvette contained in a multiple sample holder adapted to be attached to the slab gels used for the second dimensional electrophoretic separation.
Rotation of the carousel to the next station places the subject column within operating relationship to the cleaning apparatus assembly. The cleaning apparatus is used to provide cleaning solution and water to flush out and clean the inner tube in the carousel assembly after the fractions have been evacuated. The cleaning solution is preferably applied into the tube by means of a nozzle-sprayer combination that is lowered into the inner tube at the cleaning station. The cleaning solution exits from the lower part of the inner tube through the sample lines connecting the chambers to the fraction collector into a waste disposal tank.
Preferably hot air is also used at the end of the cleaning process. The cleaning process and the timing are all gated with the process sequence controls.
At this point the emptied and cleaned column is rotated back to station one in readiness to start the entire electrophoresis process over again. Alternatively, the entire carousel can be disengaged and replaced after sterilization should infectious materials, such as virus or bacteria, be in the sample media.
It should be noted the cooling system serves to provide cooling liquid to the space between the inner and outer tubes in the column assembly of the carousel. The cooling liquid, e.g. water at about 4� C., is circulated, preferably countercurrently, such as by entering through the lower port connector, then through the outer column and out through the upper port. Cooling liquid circulates through the outer column during the entire rotational cycle of the carousel assembly.
As indicated above, the specific construction and operation of the various assemblies and components of the first dimensional system are described in greater detail below.
Turning now to FIG. 2, an isometric view of the upper collector assembly of the first dimensional electrophoresis carousel is presented. This assembly provides the means for continuous fluid flow connection between the external fluid lines and the electrophoresis and cooling tubes during rotation via a stationary outlet port (not shown) which is slidably attached to the top of the upper collector. The upper collector is formed from a major cylindrical section 10 and a cylindrical section of smaller circumference 12 which is affixed onto the major section. A shelf 11 is thus formed on the top surface of section 10.
Twelve channels formed in each of three concentric ring conformations are provided in cylindrical section. The inner channel ring containing representative channel 15 is adapted to provide cooling fluid derived from an external reservoir through a stationary upper port assembly (to be described later) through the channel and then through a perpendicularly oriented channel 17 in cylindrical section 10 via bend 16. Similarly the middle channel ring containing representative channel 14 provides a buffer inlet into the upper electrode chamber via perpendicular channel 18. Finally, the outer channel ring containing representative channel 13 provides a fluid outlet for the buffer flow from the upper electrode chamber via perpendicular channel 19. Fluid flow conduits such as representative conduits 22 (linking the outer channel ring), 23 (linking the middle channel ring) and 24 (linking the inner channel ring) emanate spoke like from the upper collector assembly and provide fluid flow connection with the top inlets of the twelve electrophoresis tubes which circumferentially surround the upper collector assembly.
At other positions around the circle corresponding to different stations in the electrophoresis cycle the buffer inlet and outlet functions are replaced by fluid flow associated with fraction evacuation, column cleaning and drying. Complementary functions are provided in the lower collector assembly at the other end of the carousel assembly.
The upper collector assembly rotates with the carousel as it is mounted on the central shaft 21 which is linked to the stepping motor. Dowells 20 and 25 are provided to allow convenient mounting of the two cylindrical sections.
FIG. 3 provides several views of the components which make up the lower collector assembly and the stationary and rotating disks or surfaces which provide fluid flow control during rotation of the carousel through its cycle. Thus in FIG. 3a a view of the engaging surface of a stationary disk component is shown. This can be in the form of an independent element or can be constructed on the engaging surface of the lower port.
In FIG. 3a the stationary disk 30 is seen to be divided into a series of 4 rings of concentric holes or grooves surrounded by inert washers serving to preserve the separate fluid flow integrity of each ring. The other ring of holes such as representative holes 31, 32 and 33 provide individual connections from each column and thus represent stations where the contents of the individual electrophoresis columns are evacuated. Thus, hole 31 can be used to remove the sample containing gradient column while holes 32 and 33 can be used to pass through the wash solution and drying materials.
The next two rings represent the buffer out and buffer in rings respectively. Thus, open groove 37 will allow buffer to flow out continuously through rotation of the carousel through stations during which the electrophoresis is taking place. Similarly, open groove 38 allows buffer to flow up into the lower electrode compartment. Grooves 37 and 38 have the identical radial arc in their respective rings as they will each be open to fluid flow during the exact same positions of the rotational cycle. The innermost groove 43 allows coolant flow to pass throughout passage to all stations in the cycle.
Holes 34 and 35 in the second ring and holes 36 and 37 in the third ring are used to provide buffer cleaning to the electrode chamber at the cleaning station. Each of the rings are maintained in fluid tight relationship to each other by use of washer rings interposed between the operating rings, e.g. washer rings 41, 42 and 43. These washers can be made of any suitable inert plastic material such as Teflon.
FIG. 3b shows a view of the engaging surface of a rotating disk or surface. This disk 50 has four concentric rings of holes. Each of the rows contains twelve holes which are arranged and constructed to be in operative flow relation with the electrophoresis cycle stations and have the same orientations as the grooves and holes in the stationary ring with which they are intended to engage. Grooves 51, 52 and 53 provide a means to engage the washers.
Similarly, disk 50 can be in the form of an independent element or can be constructed on the engaging surface of the smaller cylindrical section of the lower collector.
In FIG. 3c, a cross-sectional view of the lower collector assembly is shown. Analogous to the upper collector previously discussed in FIG. 2, the lower collector comprises a major cylindrical section 60 and an affixed cylindrical section of smaller circumference 61. The fluid conduits 62, 63, 64, 65, 66 and 67 are representative of the connectors which run from the bottom inlets of the individual electrophoresis tubes and again run spoke like to the central lower collector. These channels again made a 90� bend and pass through the cylindrical section 61. Lower outlet port 68 is firmly affixed to cylindrical section 61 by means of screw cap 77. The outlet port 68 remains stationary while the lower collector assembly comprising sections 60 and 61 rotates with the entire carousel assembly of which they are an integral part.
The interface 69 between the lower collector and the outlet port is comprised of the disks shown in FIGS. 3a and 3b. The stationary disk of FIG. 3a is situated contiguous with the inner face of the outlet port 68, while the rotating disk of FIG. 3b is mounted on the inner face of cylindrical section 61 of the lower collector. The two disks or the entire assembly are made of a material such as stainless steel or precision cast plastic which allows them to be slidably engaged with each other. As the rotating disk moves circularly with the rotation of the carousel the concentric rings of holes make or break fluid flow connections between the channels of the lower port 68 and the corresponding channels in cylindrical section 61 depending on whether the holes or grooves are engaged in the stationary disk.
As shown in FIG. 3d a washer ring 70 preferably Teflon is inserted in a groove on the faces of stationary disk 71 and rotating disk 72 so as to provide complete isolation between the various rings and thus eliminate mixing of fluids or fluid loss during operation.
FIG. 4 provides a side isometric view of the carousel embodiment. The carousel body 80 is supported by brackets 81 and 98 by use of collars 82 and 99. The carousel contains twelve double tube columns arranged in circular fashion around center shaft 108 although smaller or larger numbers of such columns may be employed in alternate embodiments of the invention. A representative double tube column is shown with the electrophoresis tube 100 being supported within external cooling tube 101. Fluid flow connections between the tube electrophoresis and cooling and the central collector assemblies is provided by the ring section assembly 102 and ring section assembly 106 which in turn are connected to the upper collector 109 and the lower collector 110 by conduits 103 and 107 respectively. These conduits provide separate fluid lines for the three fluid inlets and outlets which are utilized at each station and depending on the station may include coolant in or coolant out, buffer in, buffer out, column cleaning or column drying.
Upper collector 109, which rotates with the carousel assembly, is slidably attached to upper port 104 which is stationary. Fluid flow from the six external outlet/inlet connections 84, 85, 86, 87, 88 and 89 is carried by upper fluid port 83 to the upper port 104. The fluid flow interface between the stationary upper port 104 and rotating upper collector 109 employs the disk system described in FIG. 3.
Similarly, the lower collector 110 which also rotates with the carousel assembly is slidably attached to lower port 105 which is stationary. In this instance seven external outlet/inlet connections are connected via lower fluid port 90, i.e., lines 91, 92, 93, 94, 95, 96 and 97. The functions encompassed by these outlet/inlet connections include those enumerated for the upper port plus an added line used to evacuate the contents of the electrophoresis tube.
Introduction of the gradient and the sample into the electrophoresis tube is accomplished by direct access into the tube through top hole 111 which is open to the atmosphere at stations 1 and 2 in the cycle.
FIG. 5 is a top and cross-sectional view of the ring section assembly of the upper section of the carousel showing the construction of the three ring elements. Thus the top of each outer column such as representative outer column 120 is capped by lower ring 121. The lower ring 121 has a channel 122 which provides a fluid connection from the outer column to the conduit described in FIG. 4. This channel provides an outlet for the coolant which is flowing in countercurrent fashion (from bottom to top) through outer column 120. A central open cavity 123 is provided in the lower ring to allow the inner column to be supported. Thus the lower ring is seen to have a doughnut like construction.
The outer column (and similarly the inner column) are maintained in concentric position by providing cylindrical grooves supplied with flexible gaskets 401, 402, 403 and 404 made of rubber or similar materials and by applying mechanical pressure on both sides of the columns thus making possible the assembly and disassembly of the double columns. Use of mechanical pressure also eliminates the problem of fluid leaking from the contents of the columns.
Middle ring 124 sits upon the lower ring and supports inner column 125. The inner column sits within the outer column so as to be externally cooled by the coolant flowing through the outer column during the entire electrophoresis cycle. The middle ring contains a buffer input channel 126 which is in fluid flow connection with internal chamber 127. During the electrophoresis portion of the cycle, buffer will flow continuously into this chamber. The buffer will be in fluid contact with the top of the gradient in the inner column but due to the difference in densities no substantial mixing will occur. Suitable buffer solutions will comprise mineral acids having high conductivity most preferably dilute sulfuric acid.
The upper ring 128 rests on the middle ring and provides the top electrode assembly. Electrode 129 provided preferably in the form of a hollow cylindrically shaped element and constructed out of any conventional metallic electrode material such as platinum, or paladium, and is supported within center chamber 130 which is in fluid contact with similarly disposed chamber 127. The buffer flow from these chambers outlets through buffer output channel 131. Voltage potential for the electrode is provided through conductive lead 132 which conveniently can be in the form of a metallic wire or screw. Contact with the power supply assembly is achieved through use of a spring or similar contact (not shown on the outer surface of each carousel ring) which engages conductive lead 132 throughout the rotation through the nine stations where electrophoresis takes place, a total of 240� of rotation in the cycle. The voltage employed will generally be about 1000 volts�5% and the maximum current required is approximately 20 mA per column or 180 mA for the nine tubes concurrently undergoing electrophoresis. However, as the carousel rotates the current going through the gradient in the tube is decreased. Thus, prior to reaching the evacuation station, the current through the last column may be in the order of 0.1 to 1.0 mA. Total capability of the power supply is preferably about 250 mA at 1000 volts or 250 watts.
In a further embodiment the power supply can be provided from an A.C. source of variable frequency thus allowing the carousel to be employed for dielectrophoresis. In such embodiment the columns preferably will be provided with an insert of such shape to produce a non-uniform electric field along the length of the column.
It is also possible to utilize an electromagnet as element 129 to provide a magnetic field for the separation of biological sample materials by magnetophoresis where such sample materials are labeled by magnetic microparticle in a manner known in the art.
In FIG. 6 a top and cross-sectional view of the ring section assembly of the lower section of the carousel is shown. The upper ring 133 in the lower section supports the base of the outer column 120. Coolant flow is provided into the outer column through coolant inlet channel 134. A central open cavity 135 is provided in the upper ring to allow insertion of the inner column into the outer column.
The middle ring 136 sits beneath and supports the upper ring and also serves as the base for inner column 125. Outlet channel 137 provides fluid flow connection between the internal chamber 138a of inner column 125, via chamber 138b to the external conduit and is used to evacuate the fractions at the evacuation station. A diaphragm 139 is located at the bottom of chamber 138b to provide fluid flow isolation between the sample containing gradient solution and the lower buffer as well as support for the gradient solution. This diaphragm is constructed out of materials which are permeable to ion flow but non-permeable to fluid flow. Suitable semi-permeable materials include cellulose acetate, cellophane and porous glass. In this manner ionic conductivity between the two electrodes in each column assembly is maintained.
The lower ring 140 provides support for the lower electrode or in the alternative embodiment the electromagnet 144. Voltage potential for the electrode is provided through conductive lead 145 which is in contact with the external power supply assembly in a directly analogous manner as described for the upper electrode described above. Both the buffer input channel 141 and the buffer output channel 143 are provided in the lower ring. Buffer flow is thus provided through chamber 142 and contacts electrode 144 supported therein. The construction of lower electrode 144 will be the same as described for upper electrode 129 above.
Flexible gaskets 405, 406, 407 and 408 are provided as above to facilitate assembly and disassembly and to provide a leakproof seal.
FIG. 7 provides a schematic representation of a density gradient assembly useful in the practice of the present invention. In operation, light and dense solutions (sucrose, Ficoll and the like) are placed in reservoirs 201 and 202, respectively. These solutions are withdrawn into the syringes 203 and 204 by the action of rotating cams or leadscrews 205 and 206 turning by reversible programmable stepping motors (not shown).
In the syringe filling phase two position valve 207 allows the flow of fluid from light solution reservoir 201 through line 208 and into syringe 203 when plunger 209 is withdrawn. At the same time water from reservoir 210 flows through line 211 and through the second port of valve 207, line 212, mixing device 213, line 214 and out to waste through density applicator 215 in the withdrawn position. This procedure serves to clean out the density gradient remaining in the lines from a prior column fill. Water from reservoir 210 is forced up into the system by application of air under mild pressure through line 216 under control of valve 217.
Valve 208 is shown in gradient making positions. In this position plunger 218 is pushed forward into the syringe and the contained dense solution is expelled out through valve 208, line 219 and into mixing device 213. During the filling and mixing procedures both syringes operate in tandem and in the same operating phase. Thus while dense solution is flowing from syringe 204, light solution is flowing from syringe 203. Mixing device 213 mixes the two solutions and the generated gradient is deposited into the electrophoresis column via line 214 and out through applicator 215 in the activated position. The two syringes dispensing fluid at a preprogrammable rate (i.e., unequal linear velocities), so that a linear gradient is generated.
It should be noted that at the start of density gradient generation, a small aliquot of the gradient is allowed to drain to waste by keeping the applicator in the unactivated position at the start of the gradient deposition cycle. This procedure serves to chase out any water present from the rinsing operation in the lines and mixing device. This entire process is repeated each time a new column rotates to the gradient deposition station.
FIG. 8 is a schematic representation of the sample applicator assembly. In operation, plunger 227 of syringe 220 is withdrawn thereby withdrawing sample from the sample cuvette 226 in sample holder 225 through needle 228 and valve 221 to the syringe. The sample applicator tip 224 is lowered into the density gradient containing column which has rotated to the sample deposition station.
The sample syringe plunger 227 is pushed forward expelling the sample from syringe 220 through the second port of valve 221, line 223 and then deposited above the gradient in the column through sample application tip 224. Between filling operations the application tip is lifted from the column and moves to the waste position while needle 224 is inserted into water reservoir 229 so as to wash out the system before the system handles the next sample. Such washing procedure in each cycle serves to preserve the integrity of the individual samples.
The gradient monitoring (column evacuation) assembly is shown in schematic representation in FIG. 9. When a representative electrophoretic column 300 reaches the column evacuation station, the electrophoresis is completed and the electric field is disconnected.
The buffer or electrolyte (in the isoelectric focusing mode) is then evacuated from the buffer chamber contained in the lower ring (see FIG. 6). It is desirable, particularly in the case of isoelectric focusing where the lower chamber is filled with an acid or base, to rinse the chamber with water after evacuation to ensure that the semi-permeable membrane is free of acid or base contamination. At this time automatic valve 306 opens and directs the flow of gradient out of column 304, through channel 301 in middle ring 302 of the lower section (see FIG. 6), out through line 303 and lower collector assembly 307 (shown in representational manner--see FIG. 3c for detail) where it passes through the fraction evacuation channel and leaves the rotating section and out the stationary assembly via line 308. The gradient then is passed through flow cell 309 of UV or fluorescence monitor 310 where the contents are monitored and anayzed and the output provided by electronic signal to microprocessor 311 by electronic connector 312. A second flow cell 313 lies downstream of the UV or fluorescence monitor. This second flow cell containing pH recording electrodes detects the pH of the passing gradient and it is measured by pH meter 314. The resulting signals are also fed to microprocessor 311 by electronic connector 315.
It is also possible to employ as alternate monitoring means one or more of the following: a light scattering monitor, refractive index monitor, or an electrical resistance monitor. Instrumentation for providing such monitoring are well known in the art.
Depending on the nature of the information received by the microprocessor, valve 306 is controlled to either pass the flowing gradient out through line 316 and then out to waste or upon sensing the presence of a desired component in the gradient or a preselected pH value in the gradient, the valve 306 is controlled to pass the gradient through line 318 and out through outlet tip 319 into a cuvette held by fraction collector 320.
The flow rate of column evacuation can be controlled by use of a flow controller 321 which can be either (a) a peristaltic pump, the rotational velocity of which is controlled by the microprocessor, or (b) by an adjustable flow rate restrictor of the rotational type. In the case (b) the microprocessor will control the speed of the restrictor, thus reducing the opening allowed for flow, so that a constant flow rate is obtained. This is necessary because the density gradient becomes gradually less viscous (towards the top of the column) and thus, its flow rate characteristics are accordingly increased. The microprocessor and control module will allow for the selection of various flow rates, for flexibility of application when gradients of different chemical nature are used (e.g., surose, Ficoll etc.).
The volume of liquid in line 321 between flow cells 309 and 313 is known by using loop-tubing of certain dimensions. This parameter is inputted into the microprocessor.
The microprocessor in receiving an analog signal from the pH meter and after A/D conversion will process the digital information in terms of the following:
(a) starting pH: the pH at which collection of fractions is to be initiated.
(b) terminating pH: the pH at which collection of fractions is to be terminated.
(c) pH segment: the desired pH value difference between two consecutive fractions.
(d) number of fractions to be collected (up to the maximum allowed by the fraction collector, i.e., 12�50=600).
The following mode of collection will be available:
staring pH (s)
terminating pH (t)
number of fractions to be collected (n).
Collection mode:
pH segment=(t-s)/n.
Start collecting at pH 4.0, terminate at pH 6.0, and collect 20 fractions.
Result: the fractions will differ by 0.1 pH unit.
starting pH (s)
pH segment (d)
number of fractions to be collected (n)
terminating pH=s+d n
Start collecting at pH 5.0, collect fractions differing by 0.05 pH units, collect 50 fractions.
Result: terminating pH=7.5.
Preset pH segment (d)
Result: Fractions are collected differing by d pH units starting with the first fraction.
Preset: volume of fraction
Result: Fractions of certain volume are collected regardless of pH consideration. Useful in electrophoresis and other fractionations where a pH gradient is not involved.
Other modes of operation in addition to the above are possible. For example, if one runs only six columns on the carousel, the collection can be switched to the second cassette (thus, up to 100 fractions can be collected per column, with a maximum possible of 600 for one column on the carousel).
After pH recording of the gradient flow and following the programmed microprocessor logic for each mode of operation, the following occur: (a) At the starting pH (s) the valve 306 is activated to direct the flow to the fraction collector 320 and the sample is collected. When flow cell 313 is recording (s+d) pH units the fraction collector is activated to change position to collect the next fraction. (When starting pH has not been specified, the pH of the very first volume of the gradient will be considered as starting pH), (b) the average pH and average absorbance of the first fraction is computed by the microprocessor and stored in memory, (c) these values are printed-out in tabular form with a printer and after D/A conversion are used to plot absorbance (or transmittance) and pH curves in a 2-channel recorder along with the event mark of fraction change.
The pH cell and UV cell are recording the properties of the gradient differing by certain volume, the loop 322. However, since the flow rate is constant and the volume of 302 is known, the correct (average) absorbance of a sample of certain (average) pH value can be assigned to the sample by taking this value from the microprocessor memory.
In the case samples are collected by volume, activation of the valve 306 and the fraction collector 320 will be based on microprocessor control only; because the flow rate (ml/sec) is known and the volume of each fraction (ml) is preset, the time of collection of each fraction can be estimated by the machine.
After collection of the last fraction, there is a need to rinse the tubing from collector 307 to tip 319 and to fill with water so that the flow cells will not dry out (especially the pH cell). This is performed automatically by positioning the tip of the collecting tubing 319 to the offset position 323 where there is a waste container 324. Water from inlet 325 is directed by way of the valve 326 into tubing 308 and exists into waste 324. Valve 326 after the rinsing operation is switched to the other position to allow flow of gradient from the next column.
The tip 319 is placed in the appropriate position in the fraction collector 320 to be ready for collecting the second column position. Valve 306 is placed in the closed position. The above cycle is repeated for the next column.
FIG. 10 provides a front view of the sample holder fraction collector: The tip 319 is placed in position No. 1 of the sample holder 327 and deposited in cuvette or well 328 which ends in a capillary opening 329. The sample does not leak out of the wells because of the small orifice. The fractionated samples are collected sequentially in the wells. A cover plate 330 is placed on top of holder 327 and mild pressure is applied through tubing 331 to evacuate the sample from the wells into the gel slots of the second dimension electrophoresis system.
FIG. 11 shows a front view of a volume equalizing device use in conjunction with the sample holder. Excess sample (above predetermined volume) is removed by wire needles such as 332 into reservoir 333 which is connected by means of needle 334 to mild vacuum manifold 335.
As seen in end view of the volume equalizing device in conjunction with the sample holder in FIG. 12 each well has a corresponding collecting reservoir such as 337. After sample collection, the sample collector 327 is placed on top of the second dimension polyacrylamide gel slab bearing sample slots corresponding to the position of the tips, i.e., 329.
The sample collector is then removed and electrophoresis is initiated after overlaying with buffer. Since the collected samples have higher density than the buffer, they stay at the bottom of the slots.
The excess sample collected in reservoirs of volume equalizer 333 is forced by mild pressure applied by means of manifold 337 and needle 334 to be evacuated into storage vials.
FIG. 13 is a diagrammatic view of the second dimensional system. The second dimension electrophoresis may be run on any medium conventionally employed in gel electrophoresis. A particularly preferred medium is a polyacrylamide gel which is in the form of a slab. The gel may optionally contain a small amount of a detergent such as sodium dodecyl sulfate. Such gels consist of two gel phases (a stacking gel and a separating gel) or a density gradient polyacrylamide gel both of which are articles of commerce.
To provide sample application for the second dimension the sample holder 327 containing the fractions separated during the first dimensional electrophoresis run and maintained in individual cuvettes or compartments such as 328 is attached to a gel slab such as 351 or 352 supported vertically in the tank 339. The tank will preferably support the same number of gel slabs as there are columns in the carousel preferably 12. Sample is transferred by covering the holder with and applying positive pressure to the manifold cover described in FIG. 10. Once sample transfer to the gel is completed, the second dimension electrophoresis is initiated.
The second dimensional electrophoretic process has the same power requirement as was employed in the first dimension. Thus the applied voltage will be of the same value while the total current requirements will be in about 60 mA. It is thus possible, if desired, to use the same power supply, i.e., 346 for both dimensions. Potential is applied to different sides of tank 339 by means of line 347 and electrode 348 and line 349 and electrode 350. Coolant is provided from reservoir 340 by means of pump 341, line 342 and inlet 345. The coolant surrounds the suspended slabs by countercurrent flow and exits by means of outlet 344 and line 343 where it is returned to the reservoir.
Upon completion of the electrophoresis the potential is removed from the tank and the slabs are removed. Visualization can be accomplished using procedures well known in the art. Thus the slabs may be treated with a stain selective for the materials being separated which stain can be detected colorimetrically or by fluorescence. Alternatively it is possible to utilize radiolabelled samples using radioactive isotopes and detecting the separated components by autoradiography. If the second dimensional analysis is run in a preparative mode sections of gel containing desired samples can be cut out and the sample extracted and recovered by use of appropriate solvents.
The two dimensional electrophoresis system of the present invention is primarily constructed using parts fabricated from nonreactive durable plastic, glass, synthetic rubber and stainless steel materials which are chemically resistant to all reagents currently in use in electrophoresis.
While the two dimensional system of the present invention has been described as operating as a single integrated unit it is possible to utilize individual assemblies and sub-systems as stand alone devices. In one such embodiment the carousel can be utilized in the absence of electrodes and the resulting potential field to separate biological cells by gravity sedimentation.
|Cited Patent
|Filing date
|Publication date
|Aug 13, 1973
|Feb 18, 1975
|Hoefer Scientific Instr
|Gel electrophoresis apparatus and method
|Nov 19, 1976
|Sep 13, 1977
|Hoefer Scientific Instruments
|Tube gel electrophoresis device
|Nov 24, 1976
|Dec 6, 1977
|Automatic multiple-sample applicator and electrophoresis apparatus
|Jun 21, 1976
|Nov 7, 1978
|Harald Dahms
|Method and apparatus for electrophoresis
|Nov 10, 1977
|Dec 19, 1978
|Microelectrophoretic apparatus and process
|Apr 2, 1979
|Apr 22, 1980
|Olympus Optical Co., Ltd.
|Sample feeder for use in automatic electrophoresis system
|Citing Patent
|Filing date
|Publication date
|Nov 12, 1982
|Nov 20, 1984
|Fmc Corporation
|Device for isoelectric focusing
|Feb 21, 1986
|Feb 10, 1987
|University Of Iowa Research Foundation
|Continuous rotating electrophoresis column and process of using
|Jun 26, 1986
|Apr 7, 1987
|Rhone-Poulenc Recherches
|Electrophoretic fractionation apparatus
|Mar 29, 1984
|Mar 8, 1988
|The Board Of Regents Of The University Of Nebraska
|DNA sequencing
|Apr 17, 1985
|Apr 5, 1988
|Phoresis Transfer Systems, Inc.
|Method and apparatus for separating complex mixtures of bio-organic materials
|Aug 24, 1987
|May 9, 1989
|Helena Laboratories Corporation
|Automatic pipetting apparatus
|Jun 8, 1984
|Sep 26, 1989
|Autoseq, Inc.
|Apparatus and method of analyzing nucleic acid molecules
|Jul 10, 1990
|Jan 21, 1992
|The United States Of America As Represented By The United States Department Of Energy
|Method and apparatus for continuous electrophoresis
|Jul 17, 1992
|May 9, 1995
|Beckman Instruments, Inc.
|Multi-channel automated capillary electrophoresis analyzer
|Jun 3, 1993
|May 23, 1995
|Beckman Instruments, Inc.
|Capillary and capillary retaining system
|Mar 23, 1995
|Feb 25, 1997
|Beckman Instruments, Inc.
|Capillary retaining system
|Jul 8, 1996
|May 26, 1998
|Li-Cor, Inc.
|DNA sequencing
|Aug 30, 1996
|Sep 1, 1998
|Li-Cor, Inc.
|Sequencing near infrared and infrared fluorescence labeled DNA for detecting using laser diodes and suitable labels therefor
|Dec 21, 1994
|Oct 13, 1998
|California Institute Of Technology
|Automated DNA sequencing technique
|Aug 26, 1996
|Jan 26, 1999
|The Board Of Regents Of The University Of Nebraska
|Digital DNA typing
|Nov 27, 1996
|Oct 5, 1999
|Beckman Instruments, Inc.
|Method and apparatus for displaying capillary electrophoresis data
|Jun 24, 1997
|Nov 30, 1999
|Large Scale Biology Corporation
|Automated system for two-dimensional electrophoresis
|Apr 29, 1993
|Dec 14, 1999
|Beckman Coulter, Inc.
|Automated capillary electrophoresis apparatus
|Feb 18, 1998
|Dec 21, 1999
|Li-Cor, Inc.
|DNA Sequencing
|Jul 11, 1995
|Jul 11, 2000
|Li-Cor, Inc.
|Sequencing near infrared and infrared fluorescence labeled DNA for detecting using laser diodes and suitable labels therefor
|Feb 20, 1998
|Aug 15, 2000
|Capillary electrophoresis apparatus and method
|Apr 24, 1998
|Sep 5, 2000
|Apogee Designs, Ltd.
|Electrophoresis sequencing apparatus
|Sep 27, 1999
|Sep 26, 2000
|Large Scale Biology Corporation
|Automated system for two-dimensional electrophoresis
|Jan 26, 1999
|Oct 17, 2000
|Eastman Kodak Company
|Liquid separation
|Jun 24, 1999
|Oct 24, 2000
|Large Scale Biology Corporation
|Automated system for two-dimensional electrophoresis
|Dec 11, 1995
|Nov 7, 2000
|Li-Cor, Inc.
|DNA sequencing
|Jun 7, 1995
|Mar 13, 2001
|California Institute Of Technology
|Tagged extendable primers and extension products
|Jul 21, 1995
|Mar 27, 2001
|Li-Cor, Inc.
|DNA sequencing and DNA terminators
|Jun 24, 1999
|Jun 12, 2001
|Large Scale Biology Corp.
|Automated system for two-dimensional electrophoresis
|Jun 24, 1999
|May 21, 2002
|Large Scale Biology Corporation
|Protein sample preparation for electrophoresis
|May 26, 2000
|Jun 4, 2002
|Large Scale Proteomics Corp.
|Automated system for two-dimensional electrophoresis
|Oct 6, 2000
|Jul 9, 2002
|Large Scale Proteomics Corp.
|Automated system for two-dimensional electrophoresis
|Aug 17, 2000
|Aug 20, 2002
|Large Scale Biology Corporation
|Computer-assisted methods and apparatus for identification and characterization of biomolecules
|Mar 16, 2001
|Sep 17, 2002
|Large Scale Proteomics Corp.
|Automated system for two dimensional electrophoresis
|Aug 19, 1999
|Nov 5, 2002
|Tetragen Sa
|Capillary electrophoresis apparatus having filling/refilling system and methods for use thereof
|Mar 21, 2001
|Nov 19, 2002
|Large Scale Proteomics Corp.
|Automated system for two-dimensional electrophoresis
|Aug 17, 2000
|Jan 14, 2003
|Large Scale Biology Corporation
|Two-dimensional gels for separation, identification and characterization of biomolecules
|Sep 21, 2000
|Apr 29, 2003
|Large Scale Proteomics Corporation
|Automated system for two-dimensional electrophoresis
|Apr 4, 2002
|Nov 4, 2003
|Large Scale Proteomics Corporation
|Apparatus for computer-assisted isolation and characterization of proteins
|Feb 26, 2002
|May 8, 2007
|Daniel Armstrong
|Method for separation, identification and evaluation of microbes and cells
|Jun 7, 1995
|Jan 17, 2012
|Enzo Biochem, Inc.
|Nucleic acid sequencing processes using non-radioactive detectable modified or labeled nucleotides or nucleotide analogs, and other processes for nucleic acid detection and chromosomal characterization using such non-radioactive detectable modified or labeled nucleotides or nucleotide analogs
|Oct 8, 2009
|Jan 29, 2013
|Sage Science, Inc.
|Multichannel preparative electrophoresis system
|Apr 14, 2010
|Jan 29, 2013
|Sage Science, Inc.
|Multichannel preparative electrophoresis system
|Aug 24, 2010
|Feb 4, 2014
|Samsung Electronics Co., Ltd.
|Dielectrophoresis apparatus including concentration gradient generating unit, method of separating material using the same, and method of screening condition for separating material
|Feb 27, 2014
|Mar 22, 2016
|Kimberly-Clark Worldwide, Inc.
|Method and system to clean microorganisms without chemicals
|Mar 16, 2001
|Sep 27, 2001
|Large Scale Biology Corporation
|Automated system for two dimensional electrophoresis
|Feb 26, 2002
|Oct 17, 2002
|Daniel Armstrong
|Method for separation, identification and evaluation of microbes and cells
|Mar 13, 2002
|Oct 31, 2002
|Anderson N. Leigh
|Automated system for two-dimensional electrophoresis
|Jan 31, 2005
|Jun 16, 2005
|Large Scale Biology Corporation
|Automated system for two-dimensional electrophoresis
|Jul 17, 2006
|Nov 2, 2006
|Bio-Rad Laboratories, Inc.
|Automatic electroporation optimization system
|Oct 8, 2009
|May 27, 2010
|Sage Science, Inc.
|Multichannel preparative electrophoresis system
|Apr 14, 2010
|Mar 17, 2011
|Douglas Grosvenor Sabin
|Multichannel preparative electrophoresis system
|Aug 24, 2010
|Apr 21, 2011
|Samsung Electronics Co., Ltd.
|Dielectrophoresis apparatus including concentration gradient generating unit, method of separating material using the same, and method of screening condition for separating material
|Mar 13, 2003
|Jan 10, 2012
|California Institute Of Technology
|Tagged extendable primers and extension products
|Mar 19, 1985
|Oct 9, 1985
|Board of Regents of the University of Nebraska
|DNA sequencing
|Mar 19, 1985
|Aug 3, 1988
|Board Of Regents Of The University Of Nebraska
|Dna sequencing
|Mar 27, 1986
|Oct 29, 1986
|Phoresis Transfer Systems Inc.
|Method and apparatus for separating complex mixtures of bio-organic materials
|Mar 27, 1986
|Sep 2, 1987
|Phoresis Transfer Systems Inc.
|Method and apparatus for separating complex mixtures of bio-organic materials
|Feb 13, 1989
|Sep 6, 1989
|Hitachi, Ltd.
|Apparatus for determining base sequence of nucleic acid
|Feb 13, 1989
|Sep 25, 1991
|Hitachi, Ltd.
|Apparatus for determining base sequence of nucleic acid
|Mar 14, 1989
|Nov 2, 1989
|Beckman Instruments, Inc.
|Automated capillary electrophoresis apparatus
|Mar 14, 1989
|Sep 4, 1991
|Beckman Instruments, Inc.
|Automated capillary electrophoresis apparatus
|Aug 18, 1989
|Apr 4, 1990
|Hoechst Aktiengesellschaft
|Method and apparatus for electrophoretically separating, cleaning and enriching charged or polarizable macromolecules
|Aug 18, 1989
|Sep 25, 1991
|Hoechst Aktiengesellschaft
|Method and apparatus for electrophoretically separating, cleaning and enriching charged or polarizable macromolecules
|Nov 21, 1988
|Nov 22, 1990
|Norberto Guzman
|Automated capillary electrophoresis apparatus.
|Nov 21, 1988
|Jan 22, 1992
|Norberto Guzman
|Automated capillary electrophoresis apparatus
|Apr 30, 1993
|Feb 2, 1994
|Beckman Instruments, Inc.
|Multi-channel capillary electrophoresis system
|Apr 30, 1993
|Sep 7, 1994
|Beckman Instruments Inc
|Multi-channel capillary electrophoresis system
|Jan 18, 1999
|Aug 11, 1999
|Eastman Kodak Company
|Liquid separation
|Dec 6, 2002
|Sep 29, 2004
|Bio-Rad Laboratories, Inc.
|Automatic electroporation optimization system
|Dec 6, 2002
|Dec 19, 2007
|Bio Rad Laboratories
|Automatic electroporation optimization system
|Sep 21, 1995
|May 9, 1996
|Perkin-Elmer Corporation/Applied Biosystems Division
|Rotary scanning apparatus
|Feb 19, 1999
|Aug 26, 1999
|Tetragen, S.A.
|Capillary electrophoresis apparatus and method
|Apr 26, 1999
|Nov 25, 1999
|Apogee Designs, Ltd.
|Electrophoresis sequencing apparatus
|Apr 26, 1999
|May 31, 2001
|Apogee Designs Ltd
|Electrophoresis sequencing apparatus
|Aug 18, 2000
|Mar 1, 2001
|Tetragen, S.A.
|Capillary electrophoresis apparatus having filling/refilling system and methods for use thereof
|Aug 18, 2000
|Sep 20, 2001
|Tetragen S A
|Capillary electrophoresis apparatus having filling/refilling system and methods for use thereof
|Aug 27, 2014
|May 21, 2015
|Health Diagnostic Laboratory, Inc.
|Cylinder cam for gel electropheresis
|U.S. Classification
|204/607, 204/616, 435/803
|International Classification
|Cooperative Classification
|European Classification
|Sep 8, 1983
Owner name: SEPARATION SCIENCES, INC., MASSACHUSETTS
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:CATSIMPOOLAS, NICHOLAS;REEL/FRAME:004166/0282
Effective date: 19830813
|Sep 30, 1986
|Maintenance fee reminder mailed
|Dec 4, 1986
|Fee payment
Year of fee payment: 4
|Dec 4, 1986
|Surcharge for late payment
|Oct 2, 1990
|Maintenance fee reminder mailed
|Mar 3, 1991
|Lapse for failure to pay maintenance fees
|May 14, 1991
|Expired due to failure to pay maintenance fee
Effective date: 19910303


text:
Lost Cell Phones 7Favorites [+]
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Running Time: 37 minutes
Released: 09/2008
Studio Name: Naughty Zute Girls
Series: Lost Cell Phones
Director: Mike Zute
Categories: Amateur Masturbation Gonzo All Girl
Description: Naughty Zutegirl Joselyn loses her cell phone. It's discovered by a man from the 70's who has just stepped out of a time machine. However, he is too hungover to get some action so our reward is watching Joselyn perform some fantastic nude masturbation.
3 Scenes from Lost Cell Phones 7
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Sex Acts: Modeling, Masturbation
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text:
Clad in lingerie-style bodysuit, Miley channels her inner angel and demon with her dancers and enjoys every moment of it.
Despite the on-going criticism over her sexy new image, Miley Cyrus didn't tone down her performance when hitting the stage of MTV European Music Awards over the weekend. The "Hannah Montana" star wore a cream-colored lingerie-style leotard for her set.
Accompanied with male and female dancers, Miley catered fans with her danceable single "Who Owns My Heart". She remained carefree throughout the live performance, grinding against her male and female dancers, jerking and flashing a smile at some points to show that she enjoyed her set.
"We actually came up with this concept because I wanted it to kinda be 'Angels and Demons,' but it's not a Tom Hanks reference," she previously told about the concept of her performance. "It's [about the idea] that during this world, you're always going to have temptation; basically that's kind of the whole vibe."
Beside having Miley Cyrus, the November 7 MTV EMAs also saw Linkin Park, Shakira and B.o.B performing on the stage.
Miley Cyrus:
Linkin Park:


text:
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(FREE Giveaways every Sunday and Thursday)
A tiny set, made of plastic. 1-3/8" wide x 1" tall.
My favorite part is the little insert to snap in that makes the bucket look like it's full of coal.
This item is in Dollhouse 1:12 Scale.
Rhonda did a great job of recreating an 18th century kitchen (see the photos below). More info in the blog.
- Art Projects
- Children's Book History:
- Grimms' Fairy Tales
- Mother Goose
- Graphic Design History


text:
Here some suggested organisations that offer expert advice on SN.
I feel ridiculously excited that Ds2 10 (AS) built something out of Playmobil today!(10 Posts)
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- Average
- Main course: £10
- Food spend: £25
- Opening hours
- Monday to Thursday: 11:00 - 23:00 Friday & Saturday: 11:00 - 23:30 Sunday: 11:00 - 22:30
Lovely meal with my daughter, yummy burgers and excellent service. Thankyou Byron York??
Christine Lennon
08 April 2017
from York
Byron brings proper hamburgers to the heart of York. With light flooding in from floor-to-ceiling windows, colourful leather seating and exposed brick walls make for a bright, contemporary space.
The Byron menu is made up of juicy hamburgers, served the way they should be. Properly sourced, British beef is ground fresh into 6oz patties, cooked medium (or however you fancy) so they're pink and juicy, and served in a squishy bun with a pickle on the side. Favourites include the double bacon with cheese, or the signature Byron.
Byron also offers a 'Mini Byron' kids menu, plus vegetarian options and fresh, vibrant salads. Round things off properly with fries, sides, thick-churned milkshakes, specially-selected craft beers and homemade desserts.
A family-friendly restaurant, find Byron a 12-minute walk from York Rail Station.
Byron - York
11 High Ousegate, York, YO1 8RZ
See all Byron Hamburgers
See all restaurants in North Yorkshire
See all restaurants in York
Customer reviews for Byron - York
- Food
- Value
- Ambience
- Location
- Service
- Drinks
((((( 8 April 2017 by Christine Lennon from York
Lovely meal with my daughter, yummy burgers and excellent service. Thankyou Byron York??
((((( 14 March 2017 by Debbie Horner from YORK
great onion rings and tasty burgers
((((* 22 January 2017 by Caroline from York
Yummy burger and fries. Lots of delicious things to choose including gorgeous side dishes. The bun was brioche so seemed a bit special. The onion rings were greasy and bland but our lovely waitress took the price off the bill.
((((( 11 December 2016 by Andy Botherway from Harrogate
Lovely meal, our waitress Sara was wonderful!
((((( 11 July 2016 by Juliet Vigurs-pearson from York
Met by friendly helpful staff.
Starters arrived quickly
Kids loved colouring in & drinks that looked like mockjotos.
Every detail was great service & quality excellent.
Having been to Byron in Camden the kids were excited & you didn't let them down. Great Saturday dining
((((( 27 June 2016 by Lynne Sneddon from Chester
Very friendly and efficient service. Food was delicious
((((( 26 June 2016 by Jane Anspach-sykes from York
Good food, great staff
((((( 13 June 2016 by Rachael Fisher from York
Friendly service, good choice of food, restaurant not overly busy at the time we dined.
((((* 8 June 2016 by Andrea Espada from York
We loved it, very laid back environment and no rush for us to order or pay. We loved the Byronaldo burgers and courgette fritters especially, really tasty and not too oily.
(((** 5 May 2016 by Jack from York
Service was good but had to wait a long time for a pre booked table. The food was ok at best and the drinks awful. Only one of my party finished their drink.
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I recently took 4 pregnancy test, but when I took the 5th one it came back negative. That evening I started spotting, and now I'm on my 4th day of bleeding. Could I have had an early miscarriage? I have an appointment tomorrow with my OBGYN but I'm just trying to get some mom opinions to maybe ease my mind as to what may have happened. :(
I took 4 first response which all came back positive, and 2 EPT 1 positive and 1 negative, and yes, like my regular period.
I also took one clear blue during my cycle and of course that came back negative.
This is what happened to me with my miscarriages. When I miscarried the test came out negative when I had positive ones previously. I really hope this isn't the case for you but it is a possibility. :(
Thank you ladies. I will let you all know the outcome is. I really hope this doesn't happen again :(
My fiancé and I were so excited. :(
Aww sweetie its so hard. If this is the case and you do conceive again ask your doctor about something called prometrium. I am not sure if I spelled it right. It is a horomone booster that helps prevent miscarriages. My doctor gave it to me with both of my daughters since I had two previous miscarriages. He said it wasn't a 100% proven to prevent them but it worked for almost everyone he prescribed it to. And here I am three kids later. Don't lose hope!!
Thanks Amanda :) it's been really a really sad time :( a little one would complete both of our lives. I will be asking my OB on thursday about the hormone booster. I am also making a complete lifestyle change. Eating better and also exercising more, I'm hoping to have some good news within the coming months. How long after your miscarriage did you try again?
I miscarried the end of June beginning of July. I found out in September I was pregnant again. I used the ovulation tests to help me get pregnant. The less stress and the more positives in your life the better. And that medicine really made a world of difference for me. I hope it will help you as well if your doctor thinks it will help. Its something you start like asap after you find out and take until your three month mark which is known as the safe zone but doesn't mean you can't have a miscarriage after that point. Most women just typically have them in the beginning or at the very end. You could take the medicine all the way for piece of mind but it isn't medically necessary. My doctor told me once you have hit the 3 month mark it means that it did its job and you can stop taking it. Other than a tiny bit of spotting which is normal I have never had any other complications with my pregnancies since I was prescribed that. And my last pregnancy I did not need it at all. My body just took over and did what it was supposed to.
I know it's been awhile but I'm in a similar situation. My doctor won't see me until I get another positive & just said it was most likely an early miscarriage so not much to do about it. I came to the Internet to try to find anything possible & came across your post wondering if you ever got an update about it? TIA!
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text:
- Home
- Africa
- Morocco
- Casablanca
- Telluride to Casablanca flights
Flights from Telluride (TEX) to Casablanca (CMN)Edit search
We'll show you the best deals by comparing tons of flights from Telluride to Casablanca across a wide range of airlines and travel agents. Compare the cheap flights along this route.
Travel information for Telluride to Casablanca
Currency inCasablancaد.م.1.00 = $0.11Source: Open Exchange Rates
Here are some alternative departure airports for Telluride
- Distance between Telluride and Montrose (MTJ): 42.2 miles
- Distance between Telluride and Durango (DRO): 54.9 miles
- Distance between Telluride and Cortez (CEZ): 61.4 miles
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Facts and figures about flying from Telluride to Casablanca
- Telluride to Casablanca flights have a very slim chance of being delayed
- Between Telluride and Casablanca, flights are not likely to be cancelled
- Diversions are not a regular occurrence on flights between Telluride and Casablanca


text:
- Details
DetailsVent Car Mount:~ Easy installation, just squeeze the legs of the spring steel bracket, and insert into vent. Use the 3M VHB (trademark symbol) tape if desired. Comes with rubber plug with rare earth magnet. Simply insert plug into back of GoKASE and keep it all the time on the case.
- • Easy access to tunes or GPS on the phone
- • Can plug phone charger in while phone is mounted
- • Easy on & off
- • Mount portrait or landscape
- • GoKASE not included


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Leonard Pherigo
The best way describe my voice is believing Comforting it almost draws you in my voice makes you want to be I would probably be good at advertisement all the way down to reading to a group of children
English (North American), English
Child, Middle Aged, Teen, Young Adult
Special Skills
Audiobooks, Business, Cartoons, Documentaries, Educational, Internet, Movie Trailers, Podcasting, Telephone, Television, Videogames
Well first of all I have been talking my whole life I'm really good reading sticking to the script I think I do pretty well is being an animator that's them see you guys to decide I believe my voice is unique again that will be up to y'all I hope be here when you guys say I look forward to doing business
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Hire Leonard as a voice actor. Payment is made through Voices.com SurePay, an escrow service that guarantees your work is done to your satisfaction. Send payment securely by Visa, Mastercard or PayPal through Voices.com then download your files from the website.
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Title of Your New Demo 7777 Listens
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Product Description
Impossible black and white instant square frame film for 600-type Polaroid™ cameras, with matte black frames. Images emerge in just 20 seconds, giving you a finished, fully developed picture in five minutes. The improved black and white film emulsion uses unique new chemistry. Thanks to the unique chemistry, the film now develops in normal light conditions without the need for shielding or protection from sunlight. Tones are accurate, contrast is rich and vivid, with a fresh, clear and sharp final finish.


text:
Carla – Beauty on the rocks
Released: March 22, 2014
What can make a breathtaking landscape even more exciting? A stunning girl stripping and playing with her pussy! Watch a beautiful slim brunette set up a real sex show on the rocks. Her every move is full of lure and temptation, she's so overcome by lust and desire, she knows what exactly she wants and how to get it. If you're into glamour sex movies that sparkle with sensuality and lustiness then you're gonna be impressed by this one. Every scene is totally irresistible and extremely exciting, and the perfect quality of this high definition xxx movie make it an absolute must-see.


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Centura Health has more experts in more places throughout the region. Start your health journey by finding the right one for you.
Leanne Hartford is Chief Operations Officer for Parker Adventist Hospital. She has oversite of the hospital's strategic growth as well as day-to-day operations. Leanne has been in the Adventist Health System for 20 years and has been an exemplary leader since she started at Parker Hospital in
Beginning as director of physician services, she was instrumental in successfully opening the hospital in 2004. Over the years, she has fostered growth and innovation by developing and growing numerous service lines. She has played a pivotal role in the recruitment of countless physicians of various
specialties who lead the charge in providing compassionate and personalized care to our community. Leanne also led the ambulatory development of the South Denver Group's first Neighborhood Health Center at Southlands. Leanne's passion for serving our community by meeting their health care needs continues to drive
her each and every day.
Prior to joining Parker Adventist Hospital, Leanne was with Central Texas Medical Center in San Marcos, Texas where she held various roles during her seven years of service. Her background includes extensive training in managed care contracting, negotiating, management, physician relations, and
credentialing. Leanne was a Magna Cum Laude graduate of Southwest Texas University with a Bachelor's degree in marketing.
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