User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/05/14

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May 14th, 2010

1. Run gel to verify PCR from May 11th.

-Prepare agarose gel 0.8% with 100ml TAE and 0.8g agarose, heat 4 min at potency 30, let it set.
-I used:
Ladder: 1ul
DNA dye: 1.5ul
DNA: 5ul
-Run gel 50 min at 90V, incubate with 200ul Ethidium Bromide 10 min, wash 10 min and watch it.
PCR BluePromoter1.JPG

Lanes: 1) Ladder; 2,9) DNA1 1ul; 3,10) DNA1 2ul; 4,11) DNA1 1:10 dilution; 5,12) DNA2 1ul; 6,13) DNA2 2ul; 7,14) DNA2 1:10 dilution; 8,15) Negative control; 16) DNA1; 17) DNA2; 18) Ladder & Papollo’s sample; 19-20) Papollo’s samples. 2-8 primers to amplify Blue Promoter, 86 bp; 9-15 primers to amplify OGR.

PCR from may 11th didn’t work at all!!!

2. I will do now Colony PCR, the protocol is as follows:

-Take a colony and resuspend in 200ul of Tri-EDTA 10/1-NaCl 10 mM.
-Heat 10 min at 95ºC.
-Centrifugue at 14000 rpm 2 min.
-Take 10 ul as template for PCR.
Primers for OGR were used as positive control. Unlike the PCR done on may 11th now I used 10ul of DNA because now I'm doing colony PCR. PCR mix was done as follows (ul x sample):
-Taq Polymerase 1ul
-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)