User:Eric M. Walters/Notebook/Spring 2012/2012/03/23

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Colony PCR of ΔacsA::accBCDA transformants

The transformants from 3/20 have grown up nicely, probably a couple hundred (in hurry for meeting, didn't fully count). Transformation efficiency unknown (no control), so erring on the side of screening too many. Will do 31 + negative control. X 500 μM plates are gone, using 50 μM (less selection, but won't kill like 50 mM would).

  • Each 20 μL reaction (x34 master mix)
  • GoTaq: 10 μL (340)
  • acsA-upF (100 μM): 1 μL (34)
  • acsA-dnR (100 μM): 1 μL (34)
  • H2O: 8 μL (272)

Primers are the original sites, which should now be about 20 bases outside of the homologous region Negative control also run, 7002 gDNA (should have original acsA + homologous regions, ~3.2 kb)

  • 95°C 2:00
  • 95°C :30
  • 50°C :30
  • 72°C 5:40 (5.5kb band if transformed)
    • (Cycle 30x)
  • 72°C 5:00
  • 4°C forever

Image:Annotated_colony_PCR_of_acsA;;accBCDA.png

Analysis of gel

So this is weird. I expected there to be 3.2 kb bands in the negative control and any non-transformed colonies. With the accBCDA insert, it should have been about 5.5 kb. I will check the primers I used against the accBCDA insert, as I hope that the numerous ~1 kb bands are because of a nonspecific amplification from inside the insert. I primer BLASTed the pair used against the 7002 genome and the entire insertional construct, and found no non-specific binding.


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