Team:Warsaw/Calendar-Main/16 May 2008

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PCRs for fusions

Michał K.

Gel electrophoresis (15 May PCR's) and DNA isolation from proper bands (600 bp for AID lane and 2700 bp for T7 polymerase lanes).
Electrophoresis to estimate the concentration of isolated DNA.
PCR - translation 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 16 May - AID and T7 RNA-polymerase for translation fusion
Elongation time: 4 minutes
35 cycles
Optimization of PCR - translation fusion: AID + T7 RNA-polymerase - MgCl₂ concentration and number of cycles. Fig. 2.
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
Gel electrophoresis of PCR products.

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

Fig. 2. PCR products - optimalization of MgCl₂ concentration and number of cycles: DNA ladder; 2 μl MgCl₂, 20 cycles; 3 μl MgCl₂, 20 cycles; 2 μl MgCl₂, 25 cycles; 3 μl MgCl₂, 25 cycles; 2 μl MgCl₂, 30 cycles; 3 μl MgCl₂, 30 cycles; 2 μl MgCl₂, 35 cycles; 3 μl MgCl₂, 35 cycles;