9-9-11
Picked up a new Plasmid DNA isolation kit! :)
Strains for FACS measurements grew!:)
Stickers for sequencing finally arrived!:)
Colony PCR of PBAD-cI-LVA-DT
The conventional taq polymerase tube was empty:( So I borrowed the dreamtaq from MolGen. For more infon about
dreamtaq:http://www.fermentas.com/en/products/all/modifying-enzymes/thermophilic-polymerases/ep070
Addition of MgCl2 in the mastermix will not be necessary, since the Dreamtaq buffer already contains MgCl2.
For the rest, the normal taq mastermix scheme can be used, but the amount of microliters normally used to add MgCl2 need to be
Taq 10× buffer: 40μl
dNTPs 10mM: 8μl
Forward Biobrick primer: 8μl
Reverse Biobrick primer: 8μl
Taq DNA polymerase: 2μl
MQ water: 334μl
PCR conditions:
Preheated lid: 111°C
Denaturation: 94°C for 10 min.
Cycle 33×:
denaturation: 94°C for 30s.
annealing: 60°C for 30s.
Extension: 72°C for 2,5 min.
Final extension: 72°C for 10 min.
Store infinite at 4°C
Analyse on a 1% TBE agarose gel.
Send samples for sequencing!
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