From 2013.igem.org

30 June 2013

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208 lab

Main purposes today

New PCR with x7-polymerase.

Who was in the lab

Kristian

Procedure

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

In tube 1+2+3+4+5+6 where pZA21. In 7+8+9+10+11+12 GFP SF TAT In 13+14+15+16+17+18 GFP SF Sec In 19+20+21+22+23+24 RFP

First program was 45°C annealing and 2:00 extension time with tube: 1+2, 7+8, 13+14, 19+20. Second program was ramp 68°C → 60°C annealing and 2:00 extension time with tube: 3+4, 9+10, 15+16, 21+22. Last program was a touch up program with 60°C annealing and 10 cycles with a temperature increment of 0.5°C per cycle this was looped 5 times so the final amount of annealing cycles was 50. Extension time was still 2:00 and this program was done on tube: 5+6, 11+12, 17+18, 23+24.

Purification was done by gel purification, see picture below.

Purification gel with big well number one(left uppermost) containing 1+2 next 3+4, 5+6, 7+8, 9+10, 11+12, next line from left to right; 13+17, 14+16, failed well, 21+22, 24

After all PCR-products were purified the backbone was prepared for DpnI treatment and the right amount of each of the other PCR-products were mixed. Finally the DpnI treated backbone was added USER-enzyme and BSA, mixed with the appropriate PCR-products and set for reaction.

The constructs were transformed into chemical competent E.coli right after construction and incubated 2 hours in SOC before plated on Kana plates.

Results

The gel from the days PCR. From first lane is a 100 bp ladder and thereafter the well number follows the numbers of the tubes -1. Note that tube number 15 is missing

The gel from the days PCR. From first lane is a 100 bp ladder and thereafter tube 20,21,22,23 and 24. Note that there is also primer blur in subsequent wells to the right; these are just double test wells to test previous PCR products

Conclusion from today

All PCR-products have been made and the programs for each product have been determined.