Team:Warsaw/Calendar-Main/16 May 2008

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Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7

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 - 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
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
Gel electrophoresis of PCR products.

Fig. 1. PCR products - optimization of annealing temperature: 1-DNA ladder; 2 to 8 -annealing temperature 60°C (in lane 2) to 80°C (in lane 8).

Fig. 2. PCR products - optimization 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.