Preparation of pMPMT5+AID construct
Michał K.
- Isolation of hypothetical pMPM-T5+AID plasmids from cultures inoculated on previous day.
- Restriction digest of plasmids with HindIII and NcoI (1xTango buffer) - construct control.
- Gel electrophoresis - choice of proper clones (all checked colonies). Fig. 1.
Fig. 1. 1-DNA ladder; 2 to 9-plasmids isolated from colonies of transformants and digested with HindIII and NcoI
Michał K.
- 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
- Gel electrophoresis of PCR products.
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