User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/05/11

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May 11th, 2010

1. Replate transformations from may 6th & 7th.

The transformation from may 6th has grown a few colonies and the one from may 7th has more colonies but in both cases there are some satellite collonies, I’ll replate 10 colonies from each transformation following the protocol used on april 23.

2. PCR extraction of YcgF/E promoter

a) Preparation of primers to amplify Blue Promoter from E.coli K12 genomic DNA.
b) Preparation of 15ul RNAse with 1ml of TE 10:1 pH=8 to hydrate genomic DNA extracted E. coli K12, for hydration add 20 ul of TE-RNAse and incubate for 1hr at 37ºC.
PCR mix (Final volume 50 ul):
-Buffer Taq 10x 5ul (I should’ve used 0.5 ul, not 5 ul)
-MgCl 50mM 2.5ul (can be used up to 3ul)
-dNTP’s 0.4ug/ul 2.5ul
-Primer Forward 2.5ul (can be used up to 3ul)
-Primer Reverse 2.5ul (can be used up to 3ul)
-DNA 1ul, 2ul, 1:10 dilution
I used primers for OGR as positive control.