Todays experiments

The re-restreak plates looked good this morning so I’m running 12 test colonies through PCR to see if any of them amplify the 2815bp Rec1 gene region. I poured 2 more LB +X-gal+Ampicillin plates to use as grid plates to save the colonies I checked by PCR.

My protocol was to use a toothpick to pick up a single colony form the re-restreak plates, streak out in a grid on the grid plates, and then dip the toothpick and twirl it once in 50ul PCR tubes. I used 50ul PCR reaction mixes so the colonies are even more diluted and the various cell debris won’t interfere as much with the reaction. I kept the PCR tubes on ices, and then added a 10 minute 95 degree hot start to my PCR protocol to lyse the cells. An alternative way someone suggested to do colony PCR is to diluted the colonies in TE first and then a small amount to the PCR mixture.

My controls are a No DNA control, a Positive(map 7) control, and a Negative colony PCR control – one of my re-streaks yielded blue colonies. The blue colony negative colony PCR control should not yield any product with my Rec1 primer pair.

Tomorrow I’ll run the products on a gel. If any colonies yield a 2815bp band, I’ll go back to the grid plates and do digests to confirm and check orientation. Before I do that I need to look up what cut sites are present in both the vector and the Rec1 gene product and come up with a system to make asymmetrical cuts to determine orientation as well as product size

 

I’m wrapping up the final haemophilus transformation assay.

 

12 test

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