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)