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Browse through a variety of lower and upper chest exercises designed by today's leaders in the health fitness industry. We have strength training exercises for beginner, intermediate and advanced users. These exercises will help you increase the strength and definition of your chest area. If you want more information on chest exercises or strength training of any kind, be sure to check out our Fit Facts section and Ask the Expert column. These chest exercises are also featured in full workout routines in our Workouts section.
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The Winklevoss Twins Will Play Themselves on HBO's Silicon Valley
Originally posted 03/27/2015 12:40PM
HBO's Silicon Valley is about to get real meta.
Emilia Clarke Going from Game of Thrones to Terminator Genisys
Originally posted 03/27/2015 12:10PM
She's got a British accent in real life and speaks fluent Dothraki as Daenerys Targaryan on Game of Thrones, but these days Emilia Clarke is working on a more American sound.
Gearing up to star in the upcoming movie prequel Terminator Genisys, to out this summer, Clarke says she's not new to the popular franchise.
"I was absolutely a fan. I watched Terminator growing up," Clarke told PEOPLE at this week's Game of Thrones Season 5 premiere in San Francisco.
Robert Durst: Burning Questions Answered
Originally posted 03/17/2015 09:35PM
HBO's documentary series The Jinx raised as many questions about the Robert Durst case as it answered.
Robert Durst 'Wanted to Confess,' The Jinx Producer Andrew Jarecki Says
Originally posted 03/16/2015 06:10PM
Robert Durst has long been linked to three mysterious crimes: the 1982 disappearance of his wife Kathleen, the 2000 shooting of friend Susan Berman, and the 2001 murder and dismemberment of neighbor Morris Black.
Kerry Washington to Play Anita Hill in HBO Biopic
Originally posted 03/12/2015 04:05PM
If this isn't the role of the lifetime for Kerry Washington, it sure comes close.
The Scandal star will play Anita Hill, who was thrust into the spotlight in 1991 when she accused U.S. Supreme Court nominee Clarence Thomas of sexual harassment – for an HBO biopic called Confirmation, PEOPLE has confirmed.
The movie is set to document the subsequent confirmation hearings and their indelible impact on modern culture and the workplace.
Game of Thrones: Watch Daenerys Trash Talk Everybody in the Kingdom
Originally posted 03/09/2015 02:30PM
"Lannister. Baratheon. Stark. Tyrell. They're all just spokes in a wheel."
We know how we'll be watchin' @GameOfThrones season 5.
@AppStore @HBO #HBONOW #AppleLive http://t.co/uEVwKeP9am
https://t.co/d071Y5Cglx— iTunes TV (@iTunesTV) March 9, 2015
So speaks Daenerys Targaryen (Emilia Clarke) in the latest promo for the return of Game on Thrones to HBO next month.
Along with Daenerys's vow to "break the wheel," the latest trailer promises lots of swordplay and a very scary, fire-breathing dragon. We also catch a glimpse of Jon Snow (Kit Harington) trying to rally the Wildlings.
Oh, and then there are those ominous warning signs that winter is most definitely coming.
Anna Paquin in Talks to Play Axie Muldoon in Madame X
Originally posted 03/06/2015 07:40PM
HBO's favorite vampy fairy may be returning to work on another sexy show, but this time as a mere human.
Former True Blood actress Anna Paquin is in talks to play the lead in Madame X, according to The Wrap.
The miniseries is adapted from Kate Manning's period novel My Notorious Life, which is based on a true story from the Victorian era. The series follows Axie Muldoon (Paquin), a penniless daughter of immigrants who becomes a successful, yet controversial, midwife and abortionist in New York City.
Paquin, 32, and real-life hubby Stephen Moyer, 45, are reportedly working on the project together. The duo will executive produce the series through their production company CASM Films, alongside actor-comedian Jack Black, 45, according to Deadline.
Game of Thrones Blooper Reel: What's More Deadly Than a Dragon? Stairs, Apparently (VIDEO)
Originally posted 02/23/2015 02:50PM
[YOUTUBE "y6B2ssor0wQ"] Game of Thrones may be serious, but its actors are decidedly not.
We dare you not to laugh along with the stars of the HBO series in this newly released blooper reel, which highlights season 4's biggest flubs – and falls. Because, when it comes to Westeros, stairs are apparently more dangerous than any dragon ever will be.
Among the highlights? Gwendoline Christie's laugh, Ser Pounce's on-set antics (one word: diva!) and that time Pedro Pascal almost took his own head off before the Mountain (Hafthór Júlíus Björnsson) crushed it for him.
Sarah Jessica Parker's New HBO Show's Cast Revealed
Originally posted 02/06/2015 07:00PM
Sarah Jessica Parker's got a whole new cast of girlfriends by her side.
Amanda Peet Didn't Think Game of Thrones Was a Good Idea (Also: Her Hubby's the Showrunner!)
Originally posted 01/28/2015 10:30AM
[YOUTUBE "249I4kxTBEU"] Not to cast aspersions on Amanda Peet's character, or anything, but she may not have the best judgment.
If it were up to her, Game of Thrones wouldn't have received a green light!
The actress, 43, has been married to David Benioff since 2006. He co-created GoT, now entering its fifth season, with D.B. Weiss – and Peet wasn't so sure that the fantasy series should have been adapted for television.
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subset: commoncrawl


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Nick P - 27th April 2014
It's a tidy gym.. Lots of Cycles, two rowers and loads of treadmills. The resistance machines are excellent and they have good free weights too. The staff were friendly and pleasant but they don't do towels. It's a pound for the locker.
Bernie G - 30th March 2014
Sauna wasn't working but everything else was nice
tadelech b - 30th March 2014
easy access and friendly environment


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Fans are counting down the days to this year's San Diego Comic-Con. All roads lead to SDCC Convention Center from July 18 to 21. We snooped around to find out which TV actors will show up for the fans this year. The biggest news is that "How I Met Your Mother" will be attending SDCC for the first time, with the original cast in complete attendance. Arrow, Once Upon a Time, Big Bang Theory, and Supernatural will also be there.
How I Met Your Mother - at the SDCC 2013? (There's a first time for everything!)
Josh Radnor, Cobie Smulders, Jason Segel, Neil Patrick Harris and Alyson Hannigan are joining the SDCC panel for the first time. Promoting the final season of How I Met Your Mother is bound to be a bittersweet journey for the cast and the CBS show's fans. The Hollywood Reporter notes the HIMYM folks will be showing up on July 20, Saturday. It would be fantastic if Cristin Milioti (the mother) could attend, too. Cristin, who showed up for the first time in HIMYM Season 8 finale, is the only regular cast to be added into the show.
Arrow Season 2: Who Gets to Suit Up First - Roy Harper or Black Canary?
Stephen Amell, Katie Cassidy, David Ramsey, Emily Bett Rickards and Colton Haynes will join The CW's Arrow showrunners Greg Berlanti, Marc Guggenheim and Andrew Kreisberg on the panel. Oliver Queen (Green Arrow) will soon be joined by Roy Harper and Black Canary on Arrow Season 2. You can bet a number of crazy girls will come as Black Canary in their cosplay number. The question is: Who will be the first to suit up with the Hood Guy - Katie or Colton?
Olicity shippers should not miss this cool event with the Arrow cast. THR reported the Arrow panellists will come out on July 20, too.
Once Upon a Time Season 3: Snow White, Emma Swan and Prince Charming Go to Neverland!
'Once Upon a Time' SDCC reps have yet to be named. But cast and producers will show up, according to THR. The last time we saw them, Snow White, Emma Swan and Prince Charming are all headed to Neverland to rescue Henry.
Meantime in the Enchanted Forest, Neal meets Sleeping Beauty, Prince Philip and Mulan. Who else will we seen in OUAT Season 3? Like HIMYM and Arrow, OUAT is scheduled to meet the fans on July 20.
'The Big Bang Theory' writers Steve Molaro and Bill Prady will attend, but hopes are not raised for the cast's attendance on July 19, Friday. (But Sheldon Cooper and friend live for comic-cons, right?)
'Bones' showrunners Hart Hanson and Stephen Nathan will also attend the SDCC 2013 with star David Boreanaz (Seeley Booth). They will be around on the same day as TBBT, July 19.
'Supernatural' stars Jared Padalecki, Jensen Ackles, and Misha Collins will also grace this year's comic-con. The gang's schedule falls on July 21, Sunday, the same day as 'Breaking Bad' and 'Sons of Anarchy.'
More TV Guide, Social Media Features:
Dexter Season 8 Premiere Review: The End Has Begun, Hannah McKay Still MIA; Twitterverse Sees Goodbye Posts [PHOTOS] Glee Season 5 Casting Updates: Finn and Finchel are Back; Quinn, Puck, Mercedes Not Returning? [PHOTOS] RIP Google Reader: Memes, Farewell Tweets and Tips; Feedly Says 'Happy Retirement' [PHOTOS]


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Those are so cute!! Did you make the pattern yourself or, if not, what pattern did you use? I'm just starting to learn how to knit and am half-way through making my first soaker. (I'm surprised how easy it is!) My Mom has already made one (she's been knitting for 50 yrs we figured out!) and it's super cute, she used a pattern that she used back when I was in diapers so can't remember where she got it from. I really like your longies though... I want to knit some so if you have a link to the pattern you used I'd really appreciate it if you'd share!!!
Other than that, I've bought one of each of a few different diaper brands to see how I like them before I start really buying. So far I'm really liking the Growing Green One Size fitteds -they are SO soft! I know I'm going to do fitteds with some prefolds thrown in and I'm going to try to do wool as exclusively as I can for covers. I want to knit my own mostly, but I did buy one Aristocrat to see what it was like and I'd like to check out Loveybums as well. Also thinking of having some pockets on hand for outings though.
So I still need to get my stash!!! But at least I'm making decisions, which is better than I was a month ago feeling really overwhelmed with the choices.
|Wow!! I sew, but I am just now teaching myself. The lines of my DD's dresses I make for her are so funky I am too intimidated by making diapers, especailly the sewing down eleastic part.
Diapers are GREAT for teaching yourself to sew - make the first few out of something cheap, like old t-shirts or flannel sheets, and I bet you'll be surprised just how easy it is, even the elastic! And if you use something cheap you won't feel bad if you mess it up. I actually think diapers are waaaaaaaaaaay easier to sew than things like dresses. And since I started sewing diapers I think I've actually become a much better seamstress.
Good patterns - what kind of size do you want? Free or not? Darling Diapers has a free newborn pattern, but I think the full Darling Diapers pattern is totally worth paying for - really detailed instructions, 4 different sizes, from newborn to large, and pretty much every possible option you can imagine (side closing, front closing, snaps, touchtape, covers, pockets, fitteds, whatever you want).


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1965 Oldsmobile Cutlass S Selling my 65 Olds. Mechanically very solid. Have been driving vehicle as I improve cosmetically. Rebuilt newer 455, TH 350 trans, Edelbrock headers, intake, 1406 650 cfm... More
Prior to text messaging and emailing the letters ??LOL? were notoriously known in the car business as standing for ??Little Old Lady? and was... More
JUST REDUCED!!! This is a rare 1964 Oldsmobile Jetstar Holiday Coupe. This is the same basic body and chassis of the perennial favorite Chevrolet... More
VERY COOL, 455 BIG BLOCK, AUTO TRANS, GREAT DRIVER! This is a very cool old school hot-rod styled restoration. Check out the details below: Cosmetic Paint/Body:... More
What a unique post war series American classic all original paint and upholstery.Still has the original owners manual in the glove box Rocket v-8 automatic... More
1972 Oldsmobile CutlassIf you love to tinker buy a classic car restore it turn it into a hot rodOr keep it original make it... More
1967 Oldsmobile Cutlass F85 Club CoupeOne Owner, Original, Documented Low Mileage, Well Maintained (With Receipts), Protect-O- Plate, Solid Georgia CarHave you always wanted an... More
1969 Oldsmobile 442 Holiday Coupe, Black with White stripes. Daily driver garaged kept. 455 C.U Engine,.. 400 Turbo trans with stage 2 racing kit.... More
Born the second week of June at the Lansing, Michigan plant this 1961 Oldsmobile 98 is a rare example being of only 3,804 original... More
1974 Oldsmobile Cutlass S, The original interior is in impressive condition for its age. I am the second owner and the only thing I... More
NEVADA CLASSIC CAR DEALER #40339 , VIN # 834L033767 SUPER CLEAN & RARE JETSTAR 88 2 DOOR COUPE ! RUNS AND DRIVES FANTASTIC. APPEARS TO BE... More
Holiday Coupe, Very Rare Options (Factory TAC, 4 Speed, Rear Window Defroster, A/C - which has been removed), 330 Cubic Inch, Not Original Motor, Duel Exhaust... More
ORIGINAL EQUIPMENT: + 455 cid, 8 cylinder engine, 4 barrel carburetor + Automatic transmission + Front wheel drive + Power steering & brakes + Factory AM radio with 1... More
1971 Oldsmobile 442 W-30 Convertible, 1 of only 110 W-30 Ragtops built in 1971, this car is one of only forty to get the... More
1977 OLDSMOBILE/BUICK REGAL. Built by Hutchinson-Pagan and campaigned in the former NASCAR Winston Cup Series by Tighe Scott with crew chief Harry Hyde. Finished... More
1981 Oldsmobile Toronado Brougham Coupe, Great condition. 80K miles. 5.0 liter, V8, 4 bbl, automatic transmission. Fully loaded with factory extras. Original "slat grey... More
1975 Olds Replica Covered Wagon Reproduction of a "1901" Olds. Rebuilt carbs. Briggs/Stratton Engine. Runs and drives great! Seats and tires are in good condition.... More
Nevada Classic Car Dealer # 40339, Vin # F463314 Ok kids, now for something very old skool and original.... a 1935 Olds Coupe with... More
With a 400 ci V8 engine and TH400 3-speed automatic transmission, this numbers-matching 2-door convertible is in good condition! It features gold vinyl bucket... More
For sale in our Detroit showroom is a unmistakable 1966 Oldsmobile Cutlass. This iconic 1966 Oldsmobile Cutlass has a 350-ci Rocket eight cylinder engine... More
1967 Oldsmobile Cutlass Supreme 442 for sale in our Indianapolis Showroom. This 67 442 is powered by a 425 cubic inch V8 backed by... More
For sale in our Orlando showroom is a 1962 Oldsmobile Dynamic 88. 1962 marked the year for a mild facelift in the 88 lineup... More
1978 Oldsmobile 98 Regency Sedan. Black sedan complimented by plush red velvet interior in beautiful condition overall. Period wood grain, decorative detail, and acres... More
For sale in our Detroit showroom is a sharp and mighty 1968 Oldsmobile Cutlass ram rod. This 2nd generation first model year Olds was... More
Ransom E. Olds concept of the automobile proved to be both timely and successful leading the way for American automobile pioneers development of gasoline... More
1936 Oldsmobile F36, 6 Cylinder Automatic, 213.0 cu, 3.5 L, 90 HP, 4 Door (suicide style), Original car, 79,000 Original Miles, Rear Wheel Drive, Style # 36-3619, Body # 33118, Trim # 3,... More
1970 Oldsmobile Cutlass 442 clone, 12-bolt rear end, has most 442 stuff on it, runs good, drives fine, 455 8-cyl, 400 trans, decent floors,... More
Extremely rare '1950 Oldsmobile Rocket 88 "Tin Woodie" Wagon. Vehicle looks good and runs well and is a daily driver. The wagon needs to be... More
1962 Oldsmobile 98 White exterior with burgundy interior, V-8 394 CID Hydro-matic, PS, PDB, PW, AM Radio. Only 48485 claimed miles with New tires.... More
1967 Oldsmobile Cutlass 442. Please call or email for details... More


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There are currently 437 users online. 160 members and 277 guests
Most users ever online was 947, 12-07-2013 at 00:35.
commenting for further reference.Red Langford 03-25-2015 09:22
To the top.Betweentwotrees Today, 23:05
Headed to Virgin Falls Friday night thru Sunday if anyone is interested. Hangin at Caney Fork Campsite.Major Boredom Today, 23:04
Do you need a special brand of hiking poles to go that route?rcam1977 Today, 23:03
Good luck Trevor!! If for some stupid reason you don't get picked you should consider making your own SurvivorFronkey video. Seriously, go out and doBubba Today, 23:01
Yep, have the same issue. Found a way to work with it and is no longer an issue.
I hang a little stuff sack/ditty bag from the ridge line.
Welcome from South Central NJ
There are plenty of get togethers. Feel free to stop by and check out some gear.
If you haven't made
I have a few lightweight sacks, and I was thinking about converting 2 of them into double ended for my rain fly and hammock.
Thinking about
wow thank you very much. that's what I've been looking for.sink hanger Today, 22:55


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The Family Research Council Launches Boycott Of Girl Scout Cookies Over Their Inclusion Of Transgender Children
Here's what they said in their official radio announcement. You know what this means: BUY MORE GIRL SCOUT COOKIES.
Someone else has their hand in the Girl Scouts cookie jar—and it's not who think. Hello, I'm Tony Perkins of the Family Research Council in Washington, D.C. There are plenty of politics behind that box of Thin Mints, and you should know what they are before you go buying one. As sweet as the girls are, a lot of that cookie dough goes to straight to the group's political agenda. When they aren't partnering with Planned Parenthood, they're promoting sexual 'diversity.'
Last year, the Girl Scouts decided to admit boys who dress as girls—which shouldn't come as a surprise, says FRC's Cathy Ruse, since they have a cross-dresser in their front office. And that's not all. Earlier this month, a Girl Scout employee made the mistake of stopping by the office to do extra work on her own time in a T-shirt [that said] 'Pray to End Abortion.' A supervisor ordered her to turn the shirt inside out or leave the office. She left, for good. So, says Cathy Ruse, should we all.
rightwingwatch.org / Via joemygod.blogspot.com


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Sin Ming
From Wikipedia, the free encyclopedia
|This article includes a list of references, related reading or external links, but its sources remain unclear because it lacks inline citations. (April 2013)
Sin Ming is a district in Singapore. It is situated between MacRitchie Nature Reserve and Bishan Park. It consists of both residential and industrial area.
Residential homes includes a mixture of landed properties, condominiums and Housing Development Board (HDB) blocks. Ai Tong School is situated in Sin Ming.
Like Queenstown and Leng Kee districts, Sin Ming is chock-full with auto-related industries. ComfortDelGro's Citicab and Comfort taxi companies also have their offices here. The Land Transport Authority of Singapore (LTA) has a vehicle & transit licensing division in Sin Ming.
Other amenities[edit]
Sin Ming Auto Care is a multi storey building located in Sin Ming Drive that caters to automobile repairs and products.
External links[edit]
|This Singapore-related article is a stub. You can help Wikipedia by expanding it.


text: OverviewBack to top of page Kal - MSM Plus Glucosamine Liposome Cream - 4 oz. Code#:52724 Manufacturer:Kal Size/Form:4 oz. Packaged Ship Weight:0.25 MSM (15%)PLUS GLUCOSAMINE LIPOSOME CREAM• Featuring maximum strength MSM (15%) andGlucosamine for enhanced support.• Plus, an herb base featuring Aloe Vera Gel, Boswellia Ex-tract, Devil's Claw Extract, Ginger Extract, Centella Extractand others to support healthy-looking skin.For External use only.Keep out of reach of children.


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The Wire star admits he has been plagued by a frightening dream following his portrayal of the notorious British murderer in forthcoming drama Appropriate Adult, which centres around the interviews Fred gave to cops after his arrest in 1994.
He tells Britain's Radio Times, "I have this recurring dream where I'm perched on a wall and Fred West is trying to grab me and pull me down. But I didn't let him get to me. I was determined not to."
West has also defended the film following criticism from the killer's daughter Anne Marie Davis, who claims the broadcast will cause "unimaginable distress to the families of the young girls who were murdered".
West argues, "The production team have found a way of telling this story through the eyes of a social worker who was sent to interview West in prison. I think it's effective without being sensational in any way."
The film is due to air in the U.K. later this year (11).


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Browns announce game-day improvements
BEREA, Ohio (AP)
Until they start winning, the Browns are doing all they can to keep Cleveland fans happy and entertained.
On Monday, team president Alec Scheiner unveiled a five-point plan to enhance the game-day experience for fans, starting this season. After getting feedback from fans, the team prioritized improvements to FirstEnergy Stadium. Starting during the exhibition season, fans will have easier access to the lakefront stadium, improved mobile service, upgraded team stores with more merchandise and new game-day rituals, including splashier pregame introductions, a drum line and a disc jockey on the field before games and in a booth during them.
Fans have long complained about long lines at the stadium's gates. To help alleviate the problem, the Browns have installed 20 new turnstiles and will designate 44 additional security screening chutes to quicken the flow of fans in before kickoff. Scheiner said the improvements should allow 4,000 more fans to enter every 15 minutes.
Also, the Browns are sending their season-ticket holders transparent bags to conform with the NFL's new security policy on carry bags.
''The fans have talked to us,'' Scheiner said at a news conference. ''They've been pretty clear that when they get to the stadium, it takes too long to get into the stadium. So we're going to fix that.''
The Browns' focus on improved access will start with their exhibition opener on Aug. 8 against St. Louis. Scheiner said other initiatives won't be ''rolled out'' until the regular-season opener Sept. 8 against Miami.
Scheiner, who has been with the Browns for seven months after previously working for the Dallas Cowboys, said the team has hired Contemporary Services Corporation to focus on fan safety and their overall game-day experience.
''We want to harness the energy of our fans so you think of different stadiums around the league where it's really passionate, but it's a safe environment,'' he said. ''That's what we want to replicate. I think we've got a good atmosphere for football games.''
Spotty or sometimes no cell-phone service has been a problem for the past few seasons, and the Browns believe the installation of a new tower from Verizon and an upgrade of an AT&T tower will have a significant impact.
''We think this will make a big difference for people getting on their phone, getting text messages, getting phone calls, surfing the Internet,'' he said. ''And we'll keeping looking at this and if we need more technology in the stadium then in the near future we will keep investing. ''
The Browns want to create a home-field advantage for a team that has made the playoffs just once since 1999, and reward the league's ''best fans.''
Scheiner said the hiring of Kevin Griffin, who previously worked for the Seattle Seahawks and the MLS Seattle Sounders, in a newly created position of vice president of fan experience is further proof of owner Jimmy Haslam's commitment to improve the Browns.
''We know we have the best fans,'' Scheiner said. ''We want to have the best home-field advantage so we are talking about pre-game introductions, rituals, video and audio.''
Scheiner said one of the Browns' biggest challenges is to make the experience in the stadium as satisfying for fans as if they stayed home and watched on TV.
''We have to figure out the proper balance between getting people the information that they want, that they can get at home but also making it about our game, about our team and about our product,'' he said.
Scheiner said the Cowboys experienced some of the same issues facing the Browns.
''I don't look at them as problems as much as just opportunities, and when we built the new stadium in Dallas, we had all new technology to play with and once again, we had to just revisit what it meant to be a Cowboys fan.''
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follow tom sosnoff the creator of thinkorswim on tastytrade.
follow tom sosnoff the creator of thinkorswim on tastytrade.
the logic side of finance is beautiful. the cnbc side is grotesque.
What a great sleeper network. Gonna be big one day.
Wow, these guys probably dont know much about statistics and probabilities.
Sheridan is the man!
What a breath of fresh air. Literally every financial planning stumble has been a dud, till Tastytrade.
a bibliophile
Ben Franklin is slow clapping in heaven.
because ppl can differentiate hydroponic grown from soil grown that easily.
Read the damn disclaimer you rookies. These guys get compensated to promote the stock. Then they dump it. Fuck spam
useless. please keep stumbleupon free of cancer.
For those stumbling in the dark world of finance. I provide ye a candle.
Proof of concept that probability based investing actually works.
some books are too good read, and some books make you money.
its not always about the money...
This guy saved my life from financial ruin. Thank you Tom.
Do not take investing advice from this idiot.
just want i needed. ty
The dumbest thing ever. Cash accounts at brokerages are usually free these days. This is a dumbmoney trap.


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The present invention is a method for identifying one or more characteristics of a target molecule, and comprises the steps of: converting the characteristics of the molecule into a polynucleotide of defined sequence, wherein each characteristic is represented by at least one distinct unit on the polynucleotide, the unit comprising at least a single base; contacting the polynucleotide with at least one of the nucleotides dATP, dTTP (dUTP), dGTP and dCTP, under conditions that permit the polymerisation reaction to proceed, wherein the at least one nucleotide comprises a detectable label specific for the nucleotide; removing any non-incorporated nucleotides and detecting any incorporation events; removing any labels; and repeating steps (ii) to (iv) to thereby identify the different units, and thereby the characteristics of the molecule.
1. A method for identifying a series of characteristics of a molecule comprising the steps of:
(i) converting the characteristics of the molecule into a polynucleotide of defined sequence, wherein each characteristic is represented by at least one distinct unit on the polynucleotide, the unit comprising at least a single base;
(ii) contacting the polynucleotide with at least one of the nucleotides dATP, dTTP (dUTP), dGTP and dCTP, under conditions that permit the polymerisation reaction to proceed, wherein the at least one nucleotide comprises a detectable label specific for the nucleotide;
(iii) removing any non-incorporated nucleotides and detecting any incorporation events;
(iv) removing any labels; and
(v) repeating steps (ii) to (iv) to thereby identify the different units, and thereby the characteristics of the molecule.
2. A method according to
and wherein step (ii) is carried out in the presence of nucleotides complementary to the bases of the unit but in the absence of a nucleotide complementary to that of the stop signal.
3. A method according to
4. A method according to any preceding claim, wherein each unit comprises two bases of the same type as targets for the incorporation of labelled nucleotides, with the two bases optionally separated by one or more of a third base of a different type.
5. A method according to any preceding claim, wherein the molecule is a target polynucleotide.
6. A method according to
7. A method according to any preceding claim, wherein the label is a fluorophore.
8. A method according to
9. A method according to any preceding claim, wherein the polynucleotide of step (i) is immobilised on a support material.
10. A method according to
11. A method according to any preceding claim, wherein detection is carried out by optical microscopy.
12. A method according to
13. A method for identifying one or more characteristics of a molecule comprising the steps of:
i) converting the characteristics of the molecule into a polynucleotide of defined sequence, wherein each characteristic is represented by at least one distinct unit comprising at least a two base sequence;
ii) contacting the polynucleotide with an oligonucleotide under hybridising conditions, the oligonucleotide being complementary to a unit on the polynucleotide and being detectably labelled;
iii) removing any non-hybridised oligonucleotides and detecting an hybridisation event;
iv) removing any label(s); and
v) optionally repeating steps (ii) to (iv) to thereby identify the different units, and thereby the characteristics of the target polynucleotide.
14. A method according to
15. A method according to
16. A method according to
This invention relates to methods for identifying the characteristics of molecules. In particular, the invention relates to methods for determining the sequence of a polynucleotide.
Advances in the study of molecules have been led, in part, by improvement in technologies used to characterise the molecules or their biological reactions. In particular, the study of the nucleic acids DNA and RNA has benefited from developing technologies used for sequence analysis and the study of hybridisation events.
The principal method in general use for large-scale DNA sequencing is the chain termination method. This method was first developed by Sanger and Coulson (Sanger et al., Proc. Natl. Acad. Sci. USA, 1977; 74: 5463-5467), and relies on the use of dideoxy derivatives of the four nucleotides which are incorporated into the nascent polynucleotide chain in a polymerase reaction. Upon incorporation, the dideoxy derivatives terminate the polymerase reaction and the products are then separated by gel electrophoresis and analysed to reveal the position at which the particular dideoxy derivative was incorporated into the chain.
Although this method is widely used and produces reliable results, it is recognised that it is slow, labour-intensive and expensive.
U.S. Pat. No. 5,302,509 discloses a method to sequence a polynucleotide immobilised on a solid support. The method relies on the incorporation of 3-blocked bases A, G, C and T having a different fluorescent label to the immobilised polynucleotide, in the presence of DNA polymerase. The polymerase incorporates a base complementary to the target polynucleotide, but is prevented from further addition by the 3′-blocking group. The label of the incorporated base can then be determined and the blocking group removed by chemical cleavage to allow further polymerisation to occur. However, the need to remove the blocking groups in this manner is time-consuming and must be performed with high efficiency.
WO-A-00/39333 describes a method for sequencing polynucleotide by converting the sequence of a target polynucleotide into a second polynucleotide having a defined sequence and positional information contained-therein. The sequence information of the target is said to be �magnified� in the second polynucleotide, allowing greater ease of distinguishing between the individual bases on the target molecule. This is achieved using �magnifying tags� which are predetermined nucleic acid sequences. Each of the bases adenine, cytosine, guanine and thymine on the target molecule is represented by an individual magnifying tag, converting the original target sequence into a magnified sequence. Conventional techniques may then be used to determine the order of the magnifying tags, and thereby determining the specific sequence on the target polynucleotide.
In a preferred sequencing method, each magnifying tag comprises a label, e.g. a fluorescent label, which may then be identified and used to characterise the magnifying tag.
Although the method disclosed in this patent publication has many advantages, there is still a need for improved methods for sequencing target polynucleotides.
The present invention is based on the realisation that individual characteristics of molecules can be converted into a defined polynucleotide sequence and that this defined sequence can be characterised by the incorporation of detectable labels.
According to a first aspect of the invention, a method for identifying a series of characteristics of a molecule comprises the steps of:
(i) converting the characteristics of the molecule into a polynucleotide of defined sequence, wherein each characteristic is represented by at least one distinct unit on the polynucleotide, the unit comprising at least a single base;
(ii) contacting the polynucleotide with at least one of the nucleotides dATP, dTTP (dUTP), dGTP and dCTP, under conditions that permit the polymerisation reaction to proceed, wherein the at least one nucleotide comprises a detectable label specific for the nucleotide;
(iii) removing any non-incorporated nucleotides and detecting any incorporation events;
(iv) removing any labels; and
(v) repeating steps (ii) to (iv) to thereby identify the different units, and thereby the characteristics of the molecule.
The invention permits the rapid identification of each distinct unit on the polynucleotide, which in turn allows each distinct characteristic on the target molecule to be characterised.
The method is particularly suitable for identifying one or more bases present on a target polynucleotide (molecule), for example in determining the sequence of the target polynucleotide.
In a second aspect of the invention, a method for identifying a series of characteristics of a molecule comprises the steps of:
(i) converting the characteristics of the molecule into a polynucleotide of defined sequence, wherein each characteristic is represented by at least one distinct unit comprising at least a two base sequence;
(ii) contacting the polynucleotide with an oligonucleotide under hybridising conditions, the oligonucleotide being complementary to a unit on the polynucleotide and being detectably labelled;
(iii) removing any non-hybridised oligonucleotides and detecting an hybridisation event;
(iv) removing any label(s); and
(v) optionally repeating steps (ii) to (iv) to thereby identify the different units, and thereby the characteristics of the target polynucleotide.
The present invention is described with reference to the accompanying drawings, wherein:
The invention relies on the conversion of a target molecule into a polynucleotide having distinct, defined, units of nucleic acid sequence, each unit, or unique combination of units, being representative of a particular characteristic on the target molecule.
Each unit (and hence each characteristic) can be determined by making use of the polymerase reaction to incorporate detectably-labelled complementary nucleotides onto each unit. Detecting the label for each incorporation event characterises the unit. In a preferred embodiment detailed below, the invention utilises a specific design for each unit, to permit incorporation to occur in a highly controlled manner, allowing a highly automated analysis to take place.
The term �molecule� refers to any biological or chemical molecule. Preferred molecules are biological molecules, including polynucleotides, eg DNA.
The term �polynucleotide� is well known in the art and is used to refer to a series of linked nucleic acid molecules, e.g. DNA or RNA. Nucleic acid mimics, e.g. PNA, LNA (locked nucleicacid) and 2′-O-methRNA are also within the scope of the invention.
The reference to the bases A, T(U), G and C, relates to the nucleotide bases adenine, thymine (uracil), guanine and cytosine, as will be appreciated in the art. Uracil replaces thymine when the polynucleotide is RNA, or it can be introduced into DNA using dUTP, again as well understood in the art. Similarly, reference to the nucleotides �dATP�, �dTTP�, �dUTP�, �dGTP� and �dCTP�, relates to the corresponding deoxynucleotide triphosphates, as will be evident to the skilled person.
It will be appreciated by the skilled person that base or nucleotide analogues are known and are within the scope of the present invention. The analogues retain the ability to bind (hybridise) specifically to their complement.
The polynucleotide is said to comprise distinct �units� of nucleic acid sequence. Each characteristic on the target is represented by a distinct and predefined unit, or unique combination of units. For example, if the target molecule is a polynucleotide, each base on the target polynucleotide is represented by a distinct and predefined unit. Each unit will preferably comprise two or more nucleotide bases, preferably from 2 to 50 bases, more preferably 2 to 20 bases and most preferably 4 to 10 bases, e.g. 6 bases. There are preferably at least two different bases contained in each unit. In a preferred embodiment there are three different bases in each unit. The design of the units is such that it will be possible to distinguish the different units during a �read-out� step, involving either the incorporation of detectably labelled nucleotides in a polymerisation reaction, or on hybridisation of complementary oligonucleotides. For example, each characteristic on the target is represented by a series of bases in a unit, where one base will be complementary to a labelled nucleotide introduced during the read-out step, one base will act as a �spacer� to provide separation between incorporated labels, and one base will act as a stop signal.
In a preferred embodiment, when the target molecule is a polynucleotide, two units of distinct sequence are used to represent all of the four possible bases on the target polynucleotide. According to this embodiment, the two units can be used as a binary system, with one unit representing �0� and the other representing �1�. Each base on the target is characterised by a combination of the two units. For example, adenine may be represented by �0�+�0�, cytosine by �0�+�1�, guanine by �1�+�0� and thymine by �1�+�1�, as shown in
This is demonstrated as follows:
Odd numbered template sequence:
Even numbered template sequence:
In this example, the underlined base is the target for labelled nucleotides in a polymerase reaction, the bases in parentheses are used as a stop signal, and the remaining bases are to provide separation between the labels.
In odd numbered positions (1, 3, 5, etc) the nucleotide mix, introduced during the polymerase reaction, consists of Fluor X-dUTP, Fluor Y-dCTP and dATP (dGTP is missing from the mix). The complementary base for Fluor Y is missing for �0�, and the complementary base for Fluor X is missing for �1�. Accordingly, during a polymerase reaction, if the unit �0� is present, it will be possible to detect this by monitoring for Fluor X, and if �1� is present, by monitoring for Fluor Y.
In all even numbered positions (2, 4, 6, etc) the nucleotide mix consists of the same two fluor-labelled nucleotides, but dGTP is used, not dATP, and one or more T bases define the stop signal.
After each unit has been �read� it is possible to restart the process by introducing the missing complementary nucleotide (eg. either dGTP or dATP) to allow incorporation at the stop sequence. Non-incorporated nucleotides are washed away prior to the next read-out step.
The method of the invention may be used to determine the sequence of a target polynucleotide, or may be used to identify the presence and/or type of nucleotide present at a specific position on the target polynucleotide. For example, the method may be used to identify whether specific single nucleotide polymorphisms are present on a target polynucleotide. The method may also be used for restriction mapping and haplotyping.
The different characteristics of many molecules can be dertermined using the present invention. In addition to sequencing procedures the present method may be used to identify binding characteristics of molecules, eg., protein binding properties, enzymatic properties or other chemical or biochemical property. The different characteristics may be identified by carrying out reactions to test for each characteristic and associating a specific polynucleotide unit to each molecule that undergoes reaction. For example, if the protein-binding characteristic of a molecule is to be studied, a reaction can be performed so that the molecule and a suitable protein are brought into contact under appropriate conditions and those molecules that bind to the protein are retained in a reaction compartment, and non-protein-binding molecules are removed. A specific polynucleotide unit may then be incorporated onto the molecule, to characterise the specific protein-binding property. Further binding studies using different proteins of interest may then be carried out, and subsequent binding events characterised by the sequential incorporation of polynucleotide units, to thereby form a single polynucleotide of multiple defined units.
The attachment of a polynucleotide unit onto a molecule may be carried out by various means, depending on the nature of the molecule. If the molecule is not a polynucleotide, then attachment to the molecule may be via a first linker molecule that binds to the molecule and the first polynucleotide unit. It is preferable if the attachment is via a covalent bond and so a chemical linkage is preferred. Suitable methods for binding a polynucleotide to a non-polynucleotide are known in the art.
Attachment of subsequent units can utilise base-base complementarity, so that a subsequent unit hybridises within a portion of the preceding unit and is ligated in a ligation reaction. This is described in WO-A-00/39333.
The target molecule may be converted into the defined units using methods known in the art. For example, the conversion method disclosed in WO-A-00/39333 (the content of which is incorporated herein by reference), using restriction enzymes, may be adopted. For example, if the target is a polynucleotide, the target polynucleotide may be ligated into a vector which carries a class IIS restriction site close to the point of insertion, or the target polynucleotide may be engineered to contain such a site. The appropriate class IIS restriction enzyme is then used to cleave the restriction site, resulting in an overhang in the target sequence.
Appropriate adapters which contain one or more of the units may then be used to bind to one or more of the bases of the overhang. Once the overhang of the adapter and the cleaved vector have, been hybridised, these molecules may be ligated. This will only be achieved where full complementarity along the full extent of the overhang is achieved. Blunt-end ligation may then be effected to join the other end of the adapter to the vector. By appropriate placement of a further class II restriction site (or other appropriate restriction enzyme site), which may be same or different to the previously used enzyme, cleavage may be effected such that an overhang is created in the target sequence downstream of the sequence to which the first adapter was directed. In this way, adjacent or overlapping sequences may be consecutively converted into sequences carrying the units of defined sequence.
Using this conversion system, the defined units are formed using the binary system, wherein two consecutive units are used to define a particular base on the target polynucleotide.
Having converted the target sequence into the sequential units of the second polynucleotide, the sequence of the units may then be determined, to thereby determine the target polynucleotide sequence.
This may be achieved as discussed above using the polymerase reaction to incorporate bases complementary to those on the second polynucleotide, using either selected, detectably-labelled nucleotides or nucleotides that incorporate a group for subsequent indirect labelling, and monitoring any incorporation event.
The polymerase reaction is preferably carried out under conditions that permit the controlled incorporation of complementary nucleotides one unit at a time. This enables each unit to be categorised by the detection of an incorporated label. As each unit preferably comprises a �stop� sequence, it is possible to control incorporation by supplying only those nucleotides required for incorporation onto the first unit, as described above. As each unit is recognised by a specific label, it is possible to distinguish between two different units (0 and 1) within each cycle. This enables detection of any incorporated label, and allows the identification and position of the unit to be determined.
The method may be carried out as follows:
(i) contacting the polynucleotide comprising the defined units with at least one of the nucleotides dATP, dTTP, dGTP and dCTP, under conditions that permit the polymerisation reaction to proceed, wherein the at least one nucleotide comprises a detectable label specific for that nucleotide;
(ii) removing any non-incorporated nucleotides and detecting any incorporation events;
(iii) removing the labels from incorporated nucleotide; and
(iv) repeating steps ii) to iv), to thereby identify the different units, and thereby the sequence of the target polynucleotide.
The number of different nucleotides required in step (i) of each cycle will be dependent on the design of the units. If each unit comprises only one base type, then only one nucleotide (detectably labelled) is required. However, if two bases are utilised (one as a target for the detectably labelled nucleotide and one to provide a gap between different target bases) then two nucleotides will be required (one to bind to the target base and one to �fill in� the bases between the target bases).
The use of a base as a stop signal allows the detection steps to be performed without the requirement for blocked nucleotides to prevent uncontrolled incorporation during the polymerase reaction. The stop signal is effective as the complement for the �stop� base is absent from the polymerase mix. Therefore, each unit can be characterised before a �fill-in� step is performed, using the missing nucleotide, to incorporate a complement to the stop base, which allows the next unit to be charcterised. This is carried out after the detection step. The �stop� base of one unit will not be of the same type as the first base of the subsequent unit. This ensures that the �fill-in� procedure does not progress to the next unit, Non-incorporated nucleotides used in the �fill-in� procedure can then be removed, and the next unit can then be characterised.
The choice of polymerase and detectable label will be apparent to the skilled person. The following is used as a guide only:
The preferred polymerases are Klenow Large fragment (exo-) and T4 DNA polymerase.
In a preferred embodiment, after the conversion step, multiple polynucleotides (comprising the defined units) are immobilised on a support material. This places each polynucleotide in a fixed position, and allows the sequence of each polynucleotide to be determined by aligning consecutive images of the support material to establish the order in which the labels were detected.
Polynucleotides may be attached to support materials by recognised means, including the use of biotin-avidin interactions. Methods for immobilising polynucleotides on support materials are well known in the art, and include photolithographic techniques and techniques that rely on �spotting� individual polynucleotides in defined positions on a support material. Immobilisation may also be carried out by the random distribution of polynucleotides on microbeads, nanoparticles and planar surfaces.
Immobilisation may be by specific covalent or non-covalent interactions. The interaction should be sufficient to maintain the polynucleotides on the support during washing steps to remove unwanted reaction components. Immobilisation will preferably be at either the 5′ or 3′ position, so that the polynucleotide is attached to the support at the end only. However, the polynucleotide may be attached to the support at any position along its length, the attachment acting to tether the polynucleotide to the support.
The skilled person will appreciate the appropriate means to immobilise the polynucleotide to the support material. Suitable coatings may be applied to the support to facilitate immobilisation, as will be appreciated by the skilled person. Suitable coatings include epoxy coatings (eg. 3-glycidyloxypropyltrimethoxysilane), superaldehyde coating, mercaptosilane, and isothiocyanate. Alternatively, several linker groups may be used, including PAMAM dendritic structures (Benters et al., Chem Biochem., 2001; 2: 686-694) and the immobilisation linkers described in Zhao et al., Nucleic Acids Research, 2001; 29(4): 955-959.
Suitable support materials are known in the art, and include glass slides, ceramic and silicon surfaces and plastics materials. The support is usually a flat (planar) surface.
The second polynucleotide may be immobilised on the support material to form polynucleotide arrays which may form a random or ordered pattern on the solid support. Preferably, the arrays that are used are single molecule arrays that comprise polynucieotides in distinct optically resolvable areas, e.g. as disclosed in WO-A-00/06770, the contents which incorporated herein by reference.
To carry out the polymerase reaction it will usually be necessary to first anneal a primer sequence to the polynucleotide, the primer sequence being recognised by the polymerase enzyme and acting as an initiation site for the subsequent extension of the complementary strand. The primer sequence may be added as a separate component with respect to the polynucleotide, which comprises a complementary sequence that allows the primer to anneal.
Other conditions necessary for carrying out the polymerase reaction, including temperature, pH, buffer compositions etc., will be apparent to those skilled in the art. The polymerisation step is likely to proceed for a time sufficient to allow incorporation of bases to the first unit. Non-incorporated nucleotides are then removed, for example, by subjecting the array to a washing step, and detection of the incorporated labels may then be carried out.
An alternative strategy is to use short detectably labelled oligonucleotides to hybridise to the units on the polynucleotide, and to detect any hybridisation event.
The short oligonucleotides have a sequence complementary to specific units of the polynucleotide. For example, if a binary system is used and each characteristic is defined by a different combination of units (one representing �0� and one representing �1�) the invention will require an oligonucleotide specific for the �1� unit. In this embodiment, selective hybridisation of oligonucleotides can be achieved by designing each unit to be of a different polynucleotide sequence with respect to other units. This ensures that a hybridisation event will only occur if the specific unit is present, and the detection of hybridisation events identifies the characteristics on the target molecule.
In a preferred embodiment, the label is a fluorescent moiety. Many examples of fluorophores that may be used are known in the prior art, and include:
The attachment of a suitable fluorophore to a nucleotide can be carried out by conventional means. Suitably labelled nucleotides are also available from commercial sources. The label is attached in a way that permits removal, after the detection step. This may be carried out by any conventional method, including:
i) Photobleaching
ii) Chemical bleaching
i) By antibodies raised against the fluor (eg. anti-fluorescein, anti-Oregon green)
ii) By FRET (the incorporation of a quencher next to a signal can be used to quench the signal, eg. Taqman strategy)
i) Chemical cleavage (eg. reduction of a disulfide bridge between the base and the signal)
ii) Photocleavage (eg. introduction of a nitrobenzyl or tert-butylketon group)
iii) Enzymatic (eg. α-chymotryspin digestion of peptide linker)
i) 3′-5′ Exonucleolytic degradation of filled-in nucleotides (eg. exonuclease III or by activating the 3′-5′ exonucleolytic activity of DNA polymerase when there is an absence of certain nucleotides)
i) Digestion of double-stranded DNA bearing the signal (eg. Apal, Dral, Smal sites which can be incorporated at the stop signals).
An alternative to the use of labels that permit removal, is to use inactivated labels that are reactivated during a biochemical process.
The preferred method is by photo or chemical cleavage.
When the label is a fluorophore, the fluorescent signal generated on incorporation may be measured by optical means, e.g. by a confocal microscope. Alternatively, a sensitive 2-D detector, such as a charge-coupled detector (CCD), can be used to visualise the individual signals generated, as shown in
The general set-up for optical detection is as follows:
A. Total Internal Reflection Fluorescence Microscopy (TIRFM)
B. Confocal Laser Scanning Microscopy (CLSM)
C. Two-Photon (TPLSM) and Multiphoton Laser Scanning Microscopy
The preferred methods are TIRFM and confocal microscopy.
The following Examples illustrate the invention.
Primer extension:
A target polynucleotide is converted into a series of second polynucleotides using the methods disclosed in WO-A-00/39333. Four defined second polynucleotides are used to represent 0 and 1 units in both even and odd numbered positions. The 0- and 1-units have the sequence TTTTTTACCC and TTTTTTGCCC, respectively, in odd numbered positions, while their codings are CCCCCCATTT and CCCCCCGTTT, respectively, in even numbered positions.
5′-amino labeled single-stranded second polynucleotides are generated from double-stranded template (end product of the conversion) by asymmetric PCR using 5′-amino labeled primer, DNA polymerase and dNTPs. A common primer is annealed to the amino-labeled second polynucleotides and the molecule is immobilized to an expoxy-coated glass slide via the amino-group. Conditions are chosen to avoid aggregation of the molecules (e.g. low salt) and to ensure single molecule resolution by fluorescence microscopy.
A buffer solution �odd� containing Alexa-488-dUTP (or Cy3-dUTP), Alexa-647-dCTP (or Cy5-dCTP), dATP (dGTP missing) and DNA polymerase (Klenow or T4 DNA polymerase) is added to the slides. The fluorophore labeled nucleotides contain a photocleavable linker inserted between the fluorochrome and the base. The slides are incubated for a few minutes for the polymerase reaction to occur. After a washing procedure to remove DNA polymerase and unincorporated nucleotides, a series of images covering the entire slide are captured using TIR fluorescence microscopy and ICCD-camera detection. The label is removed by photocleavage (340 nm for 2-nitrobenzyl linker), and the slide is ready for a second round after a brief wash to remove the cleaved label. A buffer solution �even� containing exactly the same constituents as used in �odd� only with dGTP replacing dATP, is added to the slide to start the fill-in of position two. Detection and removal of signal proceeds as described for cycle one. By cycling between these two buffer systems, the units are determined in a controlled manner.
Oligonucleotide Hybridisation:
The same target polynucleotide is sequenced using a method based on hybridisation. 0- and 1-units are built up from 15-20 bp sequences that define both the base on the target polynucleotide and its position. Thus, a second polynucleotide containing 40 units (i.e. 20 bp from the target polynucleotide) is built up from a repertoir of 2�40 different 15-20 bp sequences having similar melting characteristics.
5′-amino labeled single-stranded second polynucleotides are generated from double-stranded template (end product of the conversion) by asymmetric PCR using 5′-amino labeled primer, DNA polymerase and dNTPs. The second polynucleotides are immobilized to a glass slide via the amino-group, using a glass coating that can withstand several cycles of hybridization and denaturation (PAMAM dendrimer coated glass slide). Conditions are chosen to avoid aggregation of the molecules (e.g. low salt) and to ensure single molecule resolution by fluorescence microscopy.
Two different fluorophore-labeled oligonucleotides representing 0 and 1, respectively in position one are hybridised to the immobilised polynucleotides using stringent conditions (to avoid mis-hybridisation). After several stringent washes to remove unhybridised oligos, images are captured as described in Example 1.
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MISSION Our store is committed to providing the highest quality, fresh and natural food, health and wellness products. Our staff are friendly, eager to serve and ready to educate. OBJECTIVE: Provide our customers with the freshest, organically grown fruits and vegetables. Offer foods without artificial flavors, or additives. Sell earth-friendly cleansers; pure, natural supplements; and gentle, cruelty-free body care products. Support organic farms that keep our earth and water pure. PRODUCT Offer customers organic and locally grown produce, chemical- and preservative-free groceries, cruelty-free body care and eco-household products. The products are: Free of artificial preservatives. Free of chemical additives. Organically grown, whenever possible. The least processed or unadulterated version available. Non-irradiated. Cruelty free. PRICE: S.No Product Weight Vegan In The Organic Price Pink Beetroot 1. 1kg Rs.45.00 Rs.50.00 Rs.38.00 2. Cabbage 1kg Rs.40.00 Rs.45.00 Rs.40.00 3. Capsicum 1kg Rs.85.00 Rs.67.00 Rs.75.00 4. Carrots 1kg Rs.65.00 Rs.57.00 Rs.60.00 5. Cauliflower 1kg Rs.85.00 Rs.80.00 Rs.75.00 6. Ladies' Finger 1kg Rs.85.00 Rs.90.00 Rs.80.00 PRICE The prices are placed based on the cost of production . We provide the customers with the price less than the competitors by acquiring less margin, as our main motto is to "Provide our customers good health" PLACE Marketing The place we are going to choose is PLUSH market and NICHE market. In reach to the customers. Target Group Doctors. Dieticians. High income people. PLACE Production and Procuring: Transportation Nearer to the farms Distribution Channel Retail Outlet Self Vendors Production PROMOTION Direct Marketing Referential Promotion Word of Mouth COST STRUCTURE Total cost for production = Rs. 10000 Total cost for procuring = Rs. 15000 Total cost for transportation= Rs. 5000 Total cost Rs. 30000 FEASIBILITY
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Winnipeg Free Press - PRINT EDITION
Auto Club more at home at Bible study than cruise night
Slim Cessna doesn't suffer from religious guilt -- it's more like religious confusion.
"I was raised religious and I may or may not still be. Some days I am and some days I'm not," Cessna says over the phone from Fairplay, Colo.
The issue of religion comes up because for the past 20 years Cessna and his band -- Slim Cessna's Auto Club -- have offered their take on biblical tales and other stories of sin and salvation, fire and brimstone, heaven and hell, Jesus saving and times when Jesus missed the mark.
"It (the Bible) offers such good material for stories and in a way, it's a part of a tribute to the history of folk stories," Cessna says. "That's kind of what we do: they're stories and the characters in the stories are going through a wide range of things in our songs. There's something about the history of American music and the history of folk music in there. It can get pretty weird and superstitious. It's an interesting way to look and think about things.
"I'm also interested in the power of gospel music. Whether you believe in it or not, there's a power to it that's incredible and very real. Without it, rock music wouldn't be here, or not that way it is now."
The musical story of Cessna and his six-piece band is a bit all-over-the-place. Over the years the Auto Club has delved into alt-country, rockabilly, rock 'n' roll, blues and gospel.
Their latest album, Unentitled, is the band's attempt to incorporate more pop into their sound with a stronger emphasis on crafting melodies.
"It'll give it an A for effort. The most important part of that, whether we achieved it or not, was to do something different outside of our world, our understanding of what that is; whether we even understand what pop music is," says Cessna, who shares frontman duties with Jay Munly, better known as Munly Munly.
Regardless of the band's understanding of the genre, Unentitled is filled with melodic hooks that are woven seamlessly into the aural concoction the band has been perfecting for the past two decades.
Winnipeggers will get to hear what the band is preaching when Slim Cessna's Auto Club brings its famous live show to the city for the first time ever on Sunday at the West End Cultural Centre (local garage-rock/soul faves the Vibrating Beds open the show). Tickets are $18.25 at Ticketmaster and the WECC.
To celebrate the band's 20-year anniversary, the group -- which has featured more than a dozen different members over the years -- held a three-night residency at the Lion's Lair in Denver, where it formed and where it is still based, although Cessna himself calls Pittsburgh home these days.
"It is a tiny little rock club with a 110-person capacity. We had a blast -- it was the first place that ever let us play on a weekend. That was one of our early goals as a band: to play the Lion's Lair on a weekend," Cessna, 46, says with a laugh.
Despite the band's name, they were never car enthusiasts or belonged to any auto clubs popular in the Denver area.
"We used to see awesome posters for these car clubs and auto shows; before we were even playing shows we had that name. We didn't have cool cars, it was just kind of funny to use.
"My friend San Juan John had tons of cars, like Toyota pickups and Datsuns. It was all nerd cars. We weren't driving low-riders, it was more like junk and you drive whatever runs," Cessna says.
They might not have had cool vehicles, but their sound was interesting enough to catch the attention of Jello Biafra, who has released six of the band's albums on his Alternative Tentacles label.
Cessna met the former Dead Kennedys frontman through producer/Auto Club member Bob Ferbrache. Biafra and Cessna both grew up in Boulder, Colo., and during one of Biafra's trips to visit his parents he became a fan of the group after seeing a live show.
"It's an honour to be on that label. It means the world to me, still," Cessna says. "The Dead Kennedys was an important part of my life and still is. He's a guy I looked up to for years and he's a music lover. He's got discriminating tastes.
"He puts things on his label he knows will never sell because he wants to listen to it on vinyl; he doesn't listen to CDs. I think that's why he puts out our records."
Republished from the Winnipeg Free Press print edition July 26, 2012 E7
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- Kiesza, Weeknd, Arkells win multiple Junos, but Alanis Morissette steals show
- Injuries force pro hockey player to switch careers, become a country-music star
- 5 become 4: One Direction says Zayn Malik has left the group
- Local violinist earns Juno nomination
- Chris de Burgh to play Winnipeg in October
- Checking in with the 7 young actors who played the von Trapp kids in 'The Sound of Music'
- Liza Minnelli enters a substance abuse facility; rep says she's making 'excellent progress'
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One of the biggest transitions I've ever made was moving from the burbs to San Francisco. Although moving from a small city to a big city may be normal for most, it was the most eye opening experience I've ever had. Being able to experience different cultures, sites to the city, food, people has only fueled my desire and passion to travel to more destinations, whether by foot, car or plane.
Outside of traveling, I love working out, cooking, and watching movies with my wife. In addition, I'm a professional recruiter within the social media tech space and never grow tired with connecting people to their dream jobs.
Life motto: You have one life to live so live it and stop worrying! :)


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tampon exclusivité© alain wacquier - See portfolio
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["buttons","client","exclusiveness","ink","new","novelty","offering","pad","promotion","red","reserved","special","vip"]buttons client exclusiveness ink new novelty offering pad promotion red reserved special vip
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The Bath Witch
Magic for your skin, Naturally.
Natural, & Handmade soaps and lotions
for the bath and body.
Magic for your skin, Naturally.
Natural, & Handmade soaps and lotions
for the bath and body.
What's NEW with The Bath Witch:
REFER-A-Friend now going on!
Love my products? Do you tell your friends, and family to try The Bath Witch?
Make sure to tell your friends, and families to mention YOUR name to receive 10% off your next purchase, EACH TIME!
(Can not be used with other discounts, and refer a friend discounts have to be used on separate purchases)
VIP program:
For those of you who are repeat buyers, a THANK YOU to you!
after 5 purchases of $25 or more, receive $10 off your 6th purchase (redeemable through my ETSY shop)
for the Spring.
Great way to soften, and exfoliate your dry, winter skin.
Brown, sugar is a great moisturizer, and the mix of organic brown sugar, organic coconut sugar, and organic raw sugar makes a nice gentle polisher. Organic, unrefined, coconut oil, and grapeseed oil will leave your skin super silky. Mix of essential oils that smell heavenly! (scents: coconut/clove, vanilla/cinnamon, and ginger/clove.


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Jaipur - not just for the Literature Festival
Though the Jaipur Literature Festival – always colorful, often controversial -- is the premier literary event in the country, it is not all that Jaipur has to offer. Founded in 1727 by Maharaja Sawai Jai Singh II, a Kachwaha Rajput who ruled the state of Jaipur from 1699 to 1744, the city takes after his name. The Maharaja moved his capital from Amber Fort, 11 km from the city in the adjoining hills, owing to a scarcity of water and growing population. In 1853, when the Prince of Wales visited the city, the entire city was painted a shade of earthy pink to welcome him. The practice stayed, and Jaipur assumed the sobriquet of Pink City thereafter. The Nahargarh Fort, which overlooks the city from a hillock to the northwest, the Jaigarh Fort known for its enormous cannon are the other forts. The City Palace, the Prince Albert Museum, Hawa Mahal, Jantar Mantar and the Jal Mahal are some of the most-visited monuments.
Explore Jaipur through these photos.
- AUS won by 7 wkts.vs. NZAUSFinalSun, Mar 29, 2015Melbourne, AustraliaWorld Cup Coverage...
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- NZ won by 4 wkts.vs. SANZ1st Semi FinalTue, Mar 24, 2015Auckland, New ZealandWorld Cup Coverage...
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Action images from the title clash at the Melbourne Cricket Ground, March 29, 2015. [Scores] More » World Cup, Final: Australia vs New Zealand
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Grant Elliott sealed a thrilling semifinal win for New Zealand, smashing South Africa's Dale Steyn for a big six off the penultimate delivery. Elliott later consoled a devasted Steyn, who had collapsed on the pitch. More » Steyn vs Elliott: The World Cup Semifinal's Top Moment
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Thank You For Casting Your Vote!
Voting has officially closed.
Soaring into our auspicious 8th year, Greater China's most prestigious travel trade awards event , TTG China Travel Awards continues to applaud outstanding travel trade organisations in the Greater China region who have risen above the competition and delivered beyond expectations.
Award winners are determined through the opinions of the industry, which are garnered from the votes cast by travel consultants, travel experts, tour operators and destination management companies.
Winners will be announced in mid April.
The TTG China Travel Awards is a TTG Travel Trade Publishing event, and is proudly organised by TTG China, with the support of TTG-BTmice China, TTG Asia, TTGmice, TTGassociations, TTG India and TTG Asia Luxury– the region's leading travel trade resources.
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Down to earth kind of guy.
Join OkCupid
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38 • Hickory, NC • Man
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- Aug 3, 2014
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- 5′ 8″ (1.73m)
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- Mostly anything
- Smokes
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- Religion
- Atheism
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- Capricorn, but it doesn't matter
- Education
- Dropped out of two-year college
- Job
- Technology
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- $30,000–$40,000
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The Dickinson Press
Crowds flock to hear Elizabeth Smart talk about abduction
ND House committee says no to bill outlawing sexual orientation discrimination
Western leaders support oil tax formula bill, but say it won't meet all needs
Man who ran brothels reaches plea deal
Dickinson Charities shuts down bingo after 33 years, citing declining interest
More Topics
North Dakota
North Dakota's new oil train safety checks seen missing risks
Marred by recent spill, pipeline company outlines next project
Company seeks flaring exemptions: XTO Energy wants 140 wells excused from rule
Schlumberger: Prudence the new normal for U.S. oil
Manufacturers trying to find their niche in oil industry
More Topics
Jolie's message resonates with all women
Other Views: State needs a chancellor, not a manager
Shaw: Marijuana study rejection stunning
Letter: ND's pipeline integrity a good idea
Our View: Meth a real problem in western North Dakota
More Topics
April is knocking: Gather those tax form if you have not filed
Patrick Hope: Jurassic snark
Bad bets: Compulsive gambling can snare people, devastate lives
How to avoid seasonal pressure to gamble
#TBT: Early signs of the boom
More Topics
Dorothy P. Ficek
Gerald "Jerry" Schmidt
Marianna Berger
Gerald "Jerry" Schmidt
Dorothy P. Ficek
Saints down Blue Hawks
Tuesday's scoreboard
Twins' Boyer turns basement sessions with ex-pitcher Byrd into a spot on roster
Blue Hawks take two wins over Hastings, Dakota Wesleyan
Blue Hawks track gets 3 national qualifiers
More Topics
Blue Hawks
Continuing a family tradition: Woroniecki family of rural Hebron has been raising cattle, caring for the land for more than a century
Tiny Taylor experiences growth
Growing steadily: Richardton undergoing big changes as oil's sprawl drives development, growth
Envisioning the future: Hettinger Extension Center scientists try to determine what's important to producers
Elevator evolution: Technology changing how elevators like Southwest Grain operate
Weather Forecast
March 30, 2015
Letter: ND's pipeline integrity a good idea
I want to say thank you to North Dakota officials, including Gov. Jack Dalrymple, members of the Industrial Commission and the Public Service Commission for the progress we've made in improving safety of oil pipelines in our state. Their idea for creating a state-run pipeline integrity...
opinion / letters
18 hours 7 min ago
March 27, 2015
Respect the opinions and views of others
The Dickinson Wind Energy project is now public knowledge. The project surrounds the city of Taylor and includes an estimated 87 towers. With an endeavor of this magnitude, it's not surprising that attitudes about the impact and benefits sing from one end of the scale to the other....
opinion / letters
March 27, 2015 - 10:47pm
ND pipeline regulation better than federal regulation
While most of the nation rages on about the Keystone XL Pipeline, North Dakota is currently undertaking three major projects that will completely transform oil transportation in our state while adding programs to better build pipelines. When completed (in 2016, '17, and '18)...
opinion / letters
March 27, 2015 - 10:47pm
Thank you, local businesses
Often, it is the behind-the-scenes volunteers that get overlooked. As parent and leader in the District of Boy Scouts of America, I want to make sure some local area businesses don't get missed when we say "thank you" for all the behind-the-scenes work they do, which enables...
opinion / letters
March 27, 2015 - 10:46pm
March 24, 2015
Letter: Gag amendment for trafficking bill leaves women in the dark
As a student at NDSU, I take pride in our state values and support increased penalties for human trafficking and increased services to victims. As our state changes, we have to change with it — and as human trafficking becomes more of a problem, we have to step up to fight it. Senate...
opinion / letters
March 24, 2015 - 10:33pm
Letter: Why are we voting on discrimination bill?
North Dakota's state Senate defeated legislation allowing schools to decide if trained individuals could carry concealed weapons. Primary sponsor Rep. Dwight Kiefert, R-Valley City, wanted schools far from law enforcement to make LOCAL decisions. Sen. David Rust, R-Tioga, said...
opinion / letters
March 24, 2015 - 10:32pm
Letters: Republicans, governor wrong on corporate farming
The Republican legislature and the governor have just proven that a one-party system cannot work, whether Republican or Democrat. Absent any real supporting testimony, they have passed a new law which will change our state forever. The strongest anti-corporate farming law in America...
opinion / letters
March 24, 2015 - 10:30pm
March 18, 2015
Letter: Dunn County landowners trying to keep their 60 percent
I think everyone can agree that a landowner gets to decide what happens to his land, not the government. But right now, the government of Dunn County, our elected Board of Commissioners, has more influence over my land and land values than I do. And it can happen to anybody. I talked...
opinion / letters
March 18, 2015 - 10:12pm
Letter: Legislators: Do not support HCR 3047
Dear North Dakota Senate Government & Veteran Affairs Committee, It is my request that you vote "No" on House Concurrent Resolution 3047 when it comes in front of your committee. This item was, in a larger sense, voted on last November. Myself and a large majority of voters...
opinion / letters
March 18, 2015 - 10:11pm
Letter: Remove gag amendment, take stand against trafficking
The politicians who added a gag amendment to Senate Bill 2107 claim that this amendment is necessary to "prevent taxpayer funding for abortion." Senate Bill 2107 combats human trafficking and would increase the services available to victims of this terrible crime. The politicians...
opinion / letters
March 18, 2015 - 10:10pm
Ashamed of Sen. Hoeven undermining president
What was our good senator thinking? I can understand the right wingnuts of the party thinking it was a dandy idea to give "aid and comfort" to Iran, but our North Dakota "moderate" senator? Even Sen. John McCain, R-Ariz., has told Fox News that the letter was "probably"...
opinion / letters
March 18, 2015 - 10:10pm
To honor Lincoln, Republicans should support Senate bill 2279
Abraham Lincoln, a Republican, said America's "leading object" was "to afford all … a fair chance in the race of life." Surely, one's prospects in that race are shaped by his or her employment opportunities and housing options. Yet, in North Dakota, it is perfectly...
opinion / letters
March 18, 2015 - 10:09pm
March 17, 2015
Tell your senators to vote 'No' on HCR 3047
In November, the people of North Dakota wisely voted down Measure 4. House Concurrent Resolution 3011 was a proposed constitutional amendment placed on the ballot by the 2013 North Dakota Legislature. Had this measure passed, it would have severely crippled the people's right...
opinion / letters
March 17, 2015 - 6:41pm
Other views: Turn back initiative resolution
The North Dakota House concurrent resolution that would make it more difficult for North Dakotans to initiate ballot measures should be rejected in the state Senate. House Majority Leader Al Carlson, R-Fargo, introduced the resolution, which passed the House last week. Carlson's...
opinion / letters
March 17, 2015 - 6:36pm
March 15, 2015
Letter: Oil and other hazardous materials spills
What is the main reason that causes these environmental and dangerous accidents, which are preventable? Why do tanker cars leave the railroad track? If they stayed on the tracks, there would be no oil spill and no accidents. Who's responsible for the safety of and maintenance of...
opinion / letters
March 15, 2015 - 12:00am
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Wayne Christoffersenwrote a review of Reef Walled Shoe - Men's on March 2, 2015
Fancy Hipster Kicks
Familiarity: I've put it through the wringer
Fit: True to size
Walked around with these on at the Winter OR show this year and I got a few "cool shoes" remarks from some of the vendors I talked to. They are a little flat footed and don't really have room for orthotics so I wouldn't suggest wearing these for long if you don't have strong arches. Also, its pretty obvious that they were not cut from the same leather as they have colored differently as they aged. These are really a fashion over function type of buy which I don't normally do but I got a little sick of wearing my hiking boots to the bar. Bottom line, buy them then go work on your novel at the coffee shop.


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Verdantix: 45% of Global Companies to Spend More on Energy Management in 2014
Global spending on energy management by large corporations will increase or remain flat in 2014 with just 6 percent of corporations expected to spend less, according to a new survey from analyst firm Verdantix. On a worldwide basis, 45 percent of corporations will spend more on energy management in 2014 than they did in 2013.
Reflecting the downward pressure on natural gas and electricity prices and subdued economic growth, 50 percent of corporations will keep energy management spend flat.
Other key findings from The Verdantix report, "The State Of Global Corporate Energy Management" include:
- 46 percent of corporations have implemented a global energy management strategy with centralized governance while 54 percent manage decisions at a division or facility level
- Utility bill management is the most outsourced activity with 25 percent of corporations using this service; while 21 percent outsource risk management and 19 percent outsource energy data analytics
- Spend on energy management is on average 10 percent of total spend on energy procurement with significant variations by industry
- 33 percent identify compliance with legislation as the most important factor and 32 percent rank cost savings as the primary driver for spending
- Energy leaders rate CFO hurdle rates, a lack of quantifiable benefits and insufficient cost savings as the primary barriers to investing in energy management.
The data shows that energy leaders in Brazil, Germany, South Africa and the US face different risks and opportunities depending on price trends, market deregulation and investment incentives. For example, installation of on-site power generation varies enormously by country and industry.
Better energy data management is a much more entrenched spending theme. Ninety-six percent of 250 corporations believe they need to enhance the volume, quality and frequency of energy data collection and reporting. This is a shot in the arm for the diverse group of energy software vendors, including DEXMA, Envizi, IMServ, Joulex, Panoramic Power and Urjanet.
The Verdantix results come from 250 heads of energy in 13 countries and 20 industries, surveying new directions in corporate spending on renewable energy, lighting, HVAC, building technologies, energy software and energy services.
One comment on "Verdantix: 45% of Global Companies to Spend More on Energy Management in 2014"
Thanks for the DEXMA mention.
Joan P.
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The education department offers fun educational experiences for a wide range of visitors. Whether you are a family, teacher, general visitor or representative of a group, we offer programs to meet your needs and interests.
Reservations are required for all programs. There is limited space in each program so please register early. A minimum number of registrants is required to run a program.
All education programs, including Edventure Academy, camps, and overnights, will run as scheduled unless the zoo has been closed by order of Onondaga County administration. Sorry, but no refunds or rain checks will be given for 'no-shows' for any reason.
Scout groups, Rotary, home school... if you belong to, or are active in any of these or other groups and would like to arrange a custom educational program on a specific topic, call the zoo's education department. We will work with your group to design a special program to meet your needs.
For on-site programs during a field trip to the zoo, call Liz at 435-8511 x205.
For off-site Zoo to You programs, call Alicia at 435-8511 x124.
Fees, times and availability depend on the program content, duration and materials needed. When considering such a program, please plan at least 6 weeks in advance.
Wintertime in Central New York can mean snow--sometimes a lot of it! Make the most of it: gather the family, put on those mittens and enter our contest by crafting your very own snow critter!
Snow critters sculptures can be real or make-believe animals. Non-snow items can be used including clothes, sticks, stones and food coloring. Take a picture and send it to us to enter!
The winning family will score a VIP tour of one of the animal habitats at the zoo!
The top 3 snow critters will have their photos published in an upcoming MyZoo Magazine.
Contest rules:
To enter:
Deadline for submissions: 5 p.m. on Sunday, February 8, 2015
Good luck!
Rosamond Gifford Zoo • One Conservation Place, Syracuse, NY 13204 • (315) 435-8511
Joanne M. Mahoney, County Executive
© Rosamond Gifford Zoo