Michał K.:

1.Setup of 9 separate cultures from 9 colonies of transformants pMPMT5+AID (liquid LB+tetracycline).
2. Optimalization 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
3. Optimalization 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. Optimalization 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.