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Things we did today

Wetlab work

Colony PCR

PCR verification of aadA from pRL1383a using wrong conditions.
File:Rep P1 omega colony pcr.jpg
PCR verification of rep, P1 lytic regbion, and omega interposon ligation from 8-13
  • 5 colonies of rep, P1 lytic region, and aadA(pRL1383a) region, each. pSB1A3 was used as a control.
  • I changed the annealing time to 3min 30 seconds instead of the extension time, so the sites were not amplified properly. I ran the aadA(pRL1383a) construct on a gel and got a few bands, none of which were the right size. One of them should have been right though, so I am assuming this ligation did not work.
  • I ran another PCR verification on rep, P1, and the omega interposon and afterwards a gel.
  • Krystle update


  • Plasmid preps from yesterday did not yield very high quantities. I am setting up the culture for oriV1-4 and aadA(BB) 4 so I can do another plasmid prep tomorrow.
  • Inoculated for plasmid prep of GFPf and J04430 tomorrow.

Restriction Digest

  • digest of I14032 with SpeI and PstI. I want to use this to ligate to the aadA(BB) construct.
  • Checked Krystle's RE digests from last night on a gel
  • Also ran J33207 PCR product on same gel
  • Excised J33207 and GFPf bands from gel
  • RE digested:
    EtBr stained 2% agarose gel ran at 72V for 1.5 hours. Thirty microliters of the RE digests were loaded into each well. BB-pRL1383a was digested with EcoRI/PstI. Picture is mislabeled.
  • J33207, J04430, BB-pRL1383a with EcoRI and PstI
  • pRL1383a, J33207 with HindIII and BamHI (Roche, from SC)
  • BB-pRL1383a with HindIII and BamHI (from lab -20C)
  • Ran RE digests on a gel to purify segments of interest
  • Also ran RE digested B0015 from Krystle

Gel extraction

  • Used Epoch Biolabs kit to extract J33207 and GFPf DNA from agarose gel

Drylab Work


  • Verified sequence of oriT, inserted into pSB1A2. The sequences of oriT and the BioBrick sites are correct.

Oligonucleotide Design

Margaret & Grace
  • We are having trouble with TT, RBS and Promoter ligations, so we decided to synthesize these parts.
  • B0016, I14032, B0030, & B0034


  • Gel box 1 is the only one that works properly. The unlabeled gel box and box #2 may cause gels to run excessively slow, bands to blur/smear, or other weird things. Amps is really low on these boxes. All covers/power sources are good.

Quote of the Day

Happy chickens taste better. - KSGSK