Optimisation of primers from 10.06

Michał K.

1. PCR on pB30D plasmid using OmpaL_N and OmpaP_link primers (elongation length 45s).
2. PCR on pUC19 plasmid using AlphaL_link and AlphaP_XB primers (elongation length 45s).
3. PCR on pUC19 plasmid using OmegaL_link and OmegaP_EPB primers (elongation length 30s).

Each reaction was carried out with 15 and 20 cycles and in temperature gradient from 55 to 75 degrees.




Gradient PCR products: 1, 14 and 27-DNA ladder;
2 to 8 - gradient PCR for Alpha carried out with 15 cycles;
9 to 16 - gradient PCR for Alpha carried out with 20 cycles;
17 to 23 - gradient PCR for Omega carried out with 15 cycles;
24 to 31 - gradient PCR for Omega carried out with 20 cycles;
32 to 38 - gradient PCR for OmpA carried out with 15 cycles;
39 to 46 - gradient PCR for OmpA carried out with 20 cycles.
Product od each variant of PCR is in 7 lanes: 1^st is the lower border annealing temperature (55 degrees), 6^th is the higher annealing temperature (75 degrees) and 7^th is negative control without template DNA.