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

Expected results

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 preparation:
Tube Terminator T7 RBS
Water
12 µL
12 µL
Buffer 2.1
2 µL
2 µL
Plasmid pSB1C3
5 µL
5 µL
Enzyme EcoRI
0.5 µL
0.5 µL
Enzyme PstI
0.5 µL
0.5 µL

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

Expected results

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)
5 µL of the DNA mix is adding to the Master mix
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