Template:IONIS Paris/Notebook/05-06-15
Miniprep: QIAgen vs MOBIO
Miniprep test using liquid culture of Terminator T7 and RBS to check the efficiency of miniprep kit from QIAgen and MOBIO
Electrophoresis
Aim: Verification of the purification
Migration of miniprep products
Expected results
Results
Bands have more migrated than expected but it could be due to the folding of circular plasmid. Both kits are efficient.
Plasmid digestion
Tube | Terminator T7 | RBS |
---|---|---|
Water | |
|
Buffer 2.1 | |
|
Plasmid pSB1C3 | |
|
Enzyme EcoRI | |
|
Enzyme PstI | |
|
NEB double digestion
Buffer 2.1:
- EcoRI: 100%
- PstI: 75%
37°C, 1h10
Storage: -20°C
Electrophoresis
Migration of digested T7 and bJun from 29.05.2015 and digested T7 and RBS from 05.06.2015
Expected results
Results
After digestion we get expected band for T7 terminator and RBS from minipreps of the day
Gibson Assembly
Aim: first step of our VVD Biobrick
Master Mix from University of Freiburg (15µL)
Mix of DNA fragment:
- 1.5 µL pSB1C3
- 1 µL PCR.2 VVD1 (diluted 1/100)
- 2 µL PCR.2 VVD2 (diluted 1/100)
- 1 µL YC155 (diluted 1/10)
Put immediately into a hot bath at 50°C for 1 hour
Transformation
Aim: transformation of E.Coli with Gibson Assembly product
20 µL of competent cells + 5 µL of Gibson product
Protocol for bacteria transformation: using 200 µl of LB broth before incubation for 1 h at 37°C and discarding 150 µl of supernatant before plating
5 June 15