Team:Warsaw/Calendar-Main/13 May 2008

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Preparation of pMPMT5+AID construct

Michał K.

Setup of separate cultures from colonies of transformants pMPMT5+AID (liquid LB+tetracycline).

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

Michał K.

Optimization of conditions for PCR to obtain AID DNA fragment - annealing temperature gradient (from 62°C to 82°C). Fig. 1.
Primers: AIDlNrH AIDpLinB
Template DNA: pTrc99A-AID
Elongation time: 60 sec
20 cycles
Optimization of conditions for PCR - T7 RNA polymerase for translational fusion; annealing temperature gradient (from 62°C to 82°C). Fig. 2.
Primers: T7lLinkB T7pXbSal
Template DNA: E. coli Rosetta genomic DNA
Elongation time: 4 minutes
20 cycles
Optimization of conditions for PCR - T7 RNA polymerase for transcriptional fusion; annealing temperature gradient (from 62°C to 82°C). Fig. 2.
Primers: T7lRBSHi T7pXbSal
Template DNA: E. coli Rosetta and TOP10 (negative control - Fig. 3.) genomic DNA
Elongation time: 4 minutes
20 cycles
Gel electrophoresis of PCR products (there weren't any products for T7 RNA polymerase for translational fusion).

Fig. 1. Gradient PCR products for AID: 1 -DNA ladder; 2 to 13 -PCR products: In lane 2 is product of PCR reaction with annealing temperature 62°C (the lowest temperature of gradient), in lane 13 is product of PCR reaction with annealing temperature 82°C (the highest temperature of gradient). The lanes between them contain products of PCR with middle annealing temperatures.

Fig. 2. Gradient PCR products: upper - for transcriptional fusion, lower - for translational fusion; 1-DNA ladder; 2 to 12-PCR products-border annealing temperatures of gradient: 62°C (lane 2) to 82°C (lane 12)

Fig. 3. Gradient PCR products for negative control (Top10 genomic DNA): 1-DNA ladder; 2 to 6-PCR products-border annealing temperatures of gradient: 62°C (lane 2) to 82°C (lane 6).