PCRs for fusions

Michał K.:

1. Gel electrophoresis (15 May PCR's) and DNA isolation from proper bands (600 bp for AID lane and 2700 bp for T7 polymerase lanes).

2. Electrophoresis to estimate the concentration of isolated DNA.
3. PCR - translation fusion: AID + T7 RNA-polymerase - optimization (annealing temperature gradient 60°C - 80°C).

   Primers:

   AIDlNrH and T7pXbSal
   Template DNA: purified PCR products from 16 May - AID and T7 RNA-polymerase for translation fusion
   Elongation time: 4 minutes
   35 cycles
   

4. Optimization of PCR - translation fusion: AID + T7 RNA-polymerase - MgCl₂ concentration and number of cycles.

   Primers:

   AIDlNrH and T7pXbSal

   Template DNA: purified PCR products from 16 May - AID and T7 RNA-polymerase for translation fusion
   Annealing temperature: 73°C
   Elongation time: 4 minutes
   
5. Gel electrophoresis of PCR products.

PCR products - optimalization of annealing temperature: 1-DNA ladder; 2-annealing temperature 60°C, 8-annealing temperature 80°C

PCR products - optimalization of MgCl₂ concentration and number of cycles:
1-DNA ladder;
2-2μl MgCl₂, 20 cycles;
3-3μl MgCl₂, 20 cycles;
4-2μl MgCl₂, 25 cycles;
5-3μl MgCl₂, 25 cycles;
6-2μl MgCl₂, 30 cycles;
7-3μl MgCl₂, 30 cycles;
8-2μl MgCl₂, 35 cycles;
9-3μl MgCl₂, 35 cycles;