Preparation of pMPMT5+AID construct and PCRs for fusions

Michał K.:

1. Isolation of hypothetical pMPM-T5+AID plasmids from cultures inoculated on previous day.
2. Restriction digest of plasmids with HindIII and NcoI (1xTango buffer) - construct control.
3. Gel electrophoresis - choice of proper clones (all checked colonies).


1-DNA ladder; 2 to 9-plasmids isolated from 8 colonies of transformants and digested with HindIII and NcoI

4. Optimization of conditions for PCR - T7 RNA polymerase for translation fusion; annealing temperature gradient (from 62°C to 82°C); Taq polymerase with supplemented buffer (Łukasz Banasiak helped us with that problematic PCR).
   Primers: T7lLinkB T7pXbSal
   Template DNA: E. coli Rosetta genomic DNA
   Elongation time: 4 minutes
   20 cycles
   
5. Gel electrophoresis of PCR products.


PCR products. 2-annealing temperature 62°C, 6-annealing temperature 82°C