Preparation of pMPMT5+AID construct and PCRs for fusions
Michał K.
Setup of 8 separate cultures from 8 colonies of transformants pMPMT5+AID (liquid LB+tetracycline).
Optimization of conditions for PCR to obtain AID DNA fragment - annealing temperature gradient (from 62°C to 82°C).
Primers:
AIDlNrHAIDpLinB
Template DNA: pTrc99A-AID
Elongation time: 60 sec
20 cycles
1-DNA ladder; 2 to 13-PCR products (2-annealing temperature 62°C, 13-annealing temperature 82°C)
Optimization of conditions for PCR - T7 RNA polymerase for translation fusion; annealing temperature gradient (from 62°C to 82°C).
Primers:
T7lLinkBT7pXbSal
Template DNA: E. coliRosetta genomic DNA
Elongation time: 4 minutes
20 cycles
Optimization of conditions for PCR - T7 RNA polymerase for transcription fusion; annealing temperature gradient (from 62°C to 82°C).
Primers:
T7lRBSHiT7pXbSal
Template DNA: E. coliRosetta and TOP10 (negative control) genomic DNA
Elongation time: 4 minutes
20 cycles
Gel electrophoresis of PCR products (there wasn't any products for T7 RNA polymerase for translation fusion).
PCR products: upper - for transcription fusion, lower - for translation fusion; 1-DNA ladder; 2-annealing temperature 62°C; 12-annealing temperature 82°C.PCR products - negative control (Top10 genomic DNA). 2-annealing temperature 62°C, 6-annealing temperature 82°C
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1-DNA ladder; 2 to 13-PCR products (2-annealing temperature 62°C, 13-annealing temperature 82°C)
PCR products: upper - for transcription fusion, lower - for translation fusion; 1-DNA ladder; 2-annealing temperature 62°C; 12-annealing temperature 82°C.
PCR products - negative control (Top10 genomic DNA). 2-annealing temperature 62°C, 6-annealing temperature 82°C
