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).
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)
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.