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 - translational fusion: AID + T7 RNA-polymerase - optimization (annealing temperature gradient 60°C - 80°C). Fig. 1.
   Primers:
   AIDlNrH and T7pXbSal
   Template DNA: purified PCR products from 15 May - AID and T7 RNA-polymerase for translational fusion
   Elongation time: 4 minutes
   35 cycles
   
4. Optimization of PCR - translational fusion: AID + T7 RNA-polymerase - MgCl₂ concentration and number of cycles. Fig. 2.
   Primers:
   AIDlNrH and T7pXbSal
   Template DNA: purified PCR products from 15 May - AID and T7 RNA-polymerase for translational fusion
   Annealing temperature: 73°C
   Elongation time: 4 minutes
   
5. Gel electrophoresis of PCR products.