Preparation of pMPMT5+AID construct and PCRs for fusions

Michał K.:

1. Setup of 9 separate cultures from 9 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


1-DNA ladder; 2 to 13-PCR products (2-annealing temperature 62°C, 13-annealing temperature 82°C)

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
   
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
   
5. 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.