Jori
Evaluation strategies
Vessa
To Do List
- Screening 24 variants cotransformants!
- Growth ON culture
Olesja
Flowcytometry measurement of construct: PBad_GFP_DT (colony 4 & 6)
induced with 0.7 % arabinose
measurement: 0-9 h
Joyce
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!
Inoculated 50μl of the overnight culture in M9+2% casamino acids (preheated)
Let them grow for 1 h
Measure GFP expression with the FACS for PBAD-RBS-GFP-DT colonies 4 and 6
Bought taq DNA polymerase, SpeI, FastAP from the fermentas freezer after receiving the key of it from Sjoerd.
Measurements with the FACS: doing OK!
Colony 6 shows a plot shifting to the right place, showing there is GFP expression after induction with arabinose
Colony 4 also shows a plot shifting to the right place, showing there is GFP expression afterinduction with arabinose, but it
is going slower compared to colony 6...
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