From 2013.igem.org

28 June 2013

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208 lab

Main purposes today

Make PCR with x7-polymerase.

Who was in the lab

Kristian

Procedure

Made PCRs on pZA21, GFP SF TAT, GFP SF Sec, RFP all samples where made in duplicates.

In tube 1+2, 9+10 where pZA21. In 3+4, 11+12 GFP SF TAT In 5+6, 13+14, 17+18 GFP SF Sec In 7+8, 15+16 RFP

First program was 59°C annealing and 2:00 extension time with tube: 1+2, 3+4, 5+6, 7+8. Second program was 68°C annealing and 2:00 extension time with tube: 9+10, 11+12, 13+14, 15+16. Last program was 70°C annealing and 1:00 extension time with tube: 17+18.

Results

The gel from the days PCR. Note the head and trail the band leaves behind it. It looks like supercoiled plasmid DNA

The days PCR where not very successful. The gel pic. looks like that we can only see supercoiled plasmid DNA which must originate from the template DNA.

Conclusion from today

We might have used the wrong polymerase and the products we have gotten from previous PCRs with PHUSION-polymerase are without any uracil insertion.