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

iGEM UNAM-Genomics-Mexico

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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. 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)