Preparation of pMPMT5+AID construct and PCRs for fusions

Michał K.

1. Setup of 8 separate cultures from 8 colonies of transformants pMPMT5+AID (liquid LB+tetracycline).
2. Optimization of conditions for PCR to obtain AID DNA fragment - annealing temperature gradient (from 62°C to 82°C).
   Primers: AIDlNrH AIDpLinB
   Template DNA: pTrc99A-AID
   Elongation time: 60 sec
   20 cycles
Gradient PCR products: 1-DNA ladder; 2 to 13-PCR products-border annealing temperatures of gradient: 62°C (lane 2) to 82°C (lane 13)
3. Optimization of conditions for PCR - T7 RNA polymerase for translation fusion; annealing temperature gradient (from 62°C to 82°C).
   Primers: T7lLinkB T7pXbSal
   Template DNA: E. coli Rosetta genomic DNA
   Elongation time: 4 minutes
   20 cycles
   Gradient PCR products for translation fusion (only 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).
4. Optimization of conditions for PCR - T7 RNA polymerase for transcription fusion; annealing temperature gradient (from 62°C to 82°C).
   Primers: T7lRBSHi T7pXbSal
   Template DNA: E. coli Rosetta and TOP10 (negative control) genomic DNA
   Elongation time: 4 minutes
   20 cycles
   Gradient PCR products: upper - for transcription fusion, lower - for translation fusion; 1-DNA ladder; 2 to 12-PCR products-border annealing temperatures of gradient: 62°C (lane 2) to 82°C (lane 12)
5. Gel electrophoresis of PCR products (there wasn't any products for T7 RNA polymerase for translation fusion).