Gel #1, lanes 3 and 4 pertain to fragments A (160bp) and B (1100bp) in the construction file for ssb1221.
Performed digest reaction on Zymo wobble product (1 hour in 37C), then purified using Small-Frag Zymo Cleanup.
Set up final PCR reaction for SOEing.
Was not provided with enough cells (only about 100uL in tube). Added 30uL KCM to cells, and used 60uL of mixture for reaction.
Two things: 1. Picked colonies from plate into four LB tubes for growth.
2. Ran gel on SOEing product to test for product presence, ran out of time to gel purify, so have to wait until next time.
Ran gel on SOEing product... not sure if products are present.
The band is faint, and I'm not sure my SOEing product is reliable. I think it needs to be redone.
No growth in four tubes -> JCA says no red colonies on plate is problem, so redo ligation reaction (may have picked wrong vector digest). So redid ligation reaction -> transformation -> plating.
Redid A+B SOEing reaction using CA998 + G00101 and A + B fragments. Wait until tomorrow to see results.
The purified DNA was stored. Analytical gel (mapping) will be run next time.
Previously miniprepped samples were digested using the protocol described here: EcoRI/BamHI Digest of PCR Products with BamHI and BglII enzymes. The protocol is a little off - used 1 uL of plasmid DNA (since high copy number). According to analysis using ApE, the resulting digest will contain a ~2400 bp band and a ~1600 bp band (if parent vector) or ~1000 bp band if desired product vector. This will enable us to differentiate between a parent vector w/ a mutation (explaining why the colony was white) and the part desired. The plasmids were digested, but not enough time for running the gel.
The SOEing reaction was run on analytical gel and band was cut out for later Zymo gel purification.
The result from the gel was a little odd, however. It seems the ladder leaked throughout the gel, and could have contaminated my sample... I cut it (the heavier band) out nonetheless.
It looks like the correct plasmid was obtained... the second band was about 1000 bp.
Entered both samples of sb1228 into Clotho and into plates for sequencing.
Going to redo the SOEing PCR part from scratch. Produced four tubes - 2 with 33% water replaced by DMSO and 2 without, for each SOEing part.
This page was last edited on 13 March 2012, at 09:58.