lab 208

Main purpose

• Colony PCR from Pseudomonas aeruginosa (PAO) to isolate Nir.
• Colony PCR to check the presence of AMO and HAO in transormants E.coli.

Who was in the lab

Kristian, Gosia, Henrike, Julia

Procedure

Colony PCR

Colony PCR was performed according to standard method in order to check if transformed cells which grew on medium contain desired insert in pZA21 plasmid. The colonies were chosen from agar plate to be checked. Names and numbers of colonies as well as plates names and primers are:

• HAO 1-6 in pZA21 USER, primers 28a, 28b
• AMO 1-9 in pZA21 USER, primers 29a, 29b

PCR programms based on standard PCR programm with annealing temperatures 52C for HAO and 56C for AMO and extension time 3:00 mins.

Colony PCR for Nir

In order to extract Nir from Pseudomonas we used primers which do not contain uracil and new PCR programm (touch-down PCR). Phusion polymerase was used.

Names of samples are as follows: 1,2 - primers 41a, 41b - Nir, part 1 3,4 - primers 42a, 42b - Nir, part 2 5,6 - primers 11a, 11b - all fragment

PCR programm (write it later when programm will be done)

Results

Conclusion

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