IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/08/08

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Team Allergy

  • DTLs of amiRNA insert were unsuccessful. Yesterday we concluded this was because turbo cells + amp resistance don't grow on YEB media (we were out of LB+amp media). Today we must adopt a new hypothesis as 0/16 colonies grew on LB+amp media. Working theory is that the backbone digestions pictured below have the digested backbone and inserts as the two bands (top to bottom) instead of the undigested & digested backbone (in hindsight, I was trying to ligate into YFP*2, the original insert of B21). The confusion arose from the 1kb ladder, which at first appeared to match another ladder far better than 1kb (see Thursday's entry or thereabouts). This, combined with the fact that the 1kb ladder was hand-labeled, led me to suspect that it was in fact the other ladder (pR322 as pictured here). This, in turn, supported the (theorized incorrect) conclusion that the top & bottom bands here and here were undigested & digested backbone, respectively, and that the insert had diffused. I've DTL'd again, this time using the upper band (which didn't separate into digested & undigested bands after running for 30m on a .7% agarose gel) for ligation. We'll see on Monday how it went.

Team Fence


no colonies for the arp2 promoter ligations


yes colonies for ligation of exp2 and transformation of camv


File:86 digests.jpg

  • ladder
  • 1. pACT2 (backbone)
  • 2. RxRLC (insert)
  • 3. Barstar (insert)
  • 4. Barnase (insert)
  • 5. 5XGalUAS (backbone)
  • 6. PENTcup (backbone)
  • 7. V26 (backbone)
  • 8. Ecr-Galv (insert)
        • no insert for 2 (RxRLC) or 8 (Ecr-Galv)

File:88 other digests.jpg

  • ladder
  • RxRLC (Xba and Pst) (insert)
  • RxRLC (Bam and Xho) (insert) NO INSERT HERE
  • PhyB (backbone)
  • V24 (backbone)
  • VP16 (backbone)