Virginia Commonwealth/13 August 2009

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Thursday 13 August 2009

Results

Maria and Afton

• A Gel Electrophoresis was run yesterday
  • Results show the promoters were digested properly, although only one part of the digest was visible. This was likely caused frm the gel running too long, and as a result the pieces ran off of the gel.
  • The backbone and RET digested 8/11 was not digested properly. However the digests done on those parts on 8/12 looked good.
  • Also the Miniprep of J23101 looked odd. There were a lot of pieces there instead of nice clean bands
• The gel was run on the following DNA

• Results of the Gel Electrophoresis

from left to right: J23100, J23102, J23103, J23104, J23105, J23101 (Miniprep), J23101 (Digest), J06702 (8/12), J06702 (8/11), J06702 (Miniprep), pSB1C3 (8/12), pSB1C3 (Miniprep), Low Molecular Weight Ladder, Bioline Hyperladder

Trentay 20:05, 13 August 2009 (UTC)

Tasks

• Run PCR reaction Bussingkm 15:42, 13 August 2009 (UTC)

Maria and Afton

• Ligate promoter Digests (100-107) with RET and backbone digested 8/12/09
  • They will be frozen until cuvettes come in
  • Only Digests of pSB1C3 and J06702 made on 8/12 were used in today's ligations
• Parts cannot be transformed until new cuvettes come in (they have been ordered)
• Update Notebook and Wiki
• Make cell stock of I1352 w/ pSB1C3
• Only 8 samples of NEB10β are left in the -80 degree Celsius freezer, so plans should be made to make more next week.
• Promoter Miniprepped DNA is getting low on some promoters like 100 and 101, so overnight cultures will be made of these two
  • Most of the promoter DNA has enough for another to to three digestions so it should be enough; it just seems to be getting low

Trentay 16:27, 13 August 2009 (UTC)

Wetlab

• The purified PCR product from yesterday's reaction did not show up on a 2% agarose gel next to Hyperlader IV. The 100 BP line of the ladder was visible but the product was not. Either the purification was bad or the results ran off the gel. I think there was an error in purification. I will re-do the PCR reaction of F and R primers onto promoter designs 1-9. I will first optimize Mg2+ concentration with D9+F+R. All reagents will be frozen and kept on ICE as much as possible. I will be using the PCR protocol in the protocol section and will be using Taq mixture for the reaction. A gel with each sample from D9 will be run to confirm the reaction. The expected length of D9 is 148 bp. I will use a 100 bp ladder. Bussingkm 15:42, 13 August 2009 (UTC)

From left to right: D9 (1.5 uL Mg2+), D9 (1.0 uL Mg2+), D9 (0.5 uL Mg2+), D9 (0 uL Mg2+), 100 bp ladder

The gel shows that 1.0 uL Mg2+ is optimal for this reaction. Promoters D1-8 were then mixed using the same protocol and 1.0 uL Mg2+. A gel was immediately run to confirm the PCR product.

Maria and Afton

• Ligation was done
• Overnight cultures were made of J23100 and J23101 for a Miniprep tomorrow
  • ~10µL of DNA was placed in 5mL of LB+Amp broth

Trentay 21:31, 13 August 2009 (UTC)

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