Template:IONIS Paris/Notebook/22-07-15

Results of bacterial transformation


A total of 12 colonies in both plates containing transformed bacteria
No colony into the negative control
“Biofilm” into the positive control, bacteria are alive and competent

Gel extraction from the cut band of the 16th of July


We used the kit from QIAgen to extract the DNA from the gel
Final elution into 50 µL

Digestion of Gibson product (VVD-YC155)

Mix for digestion
Tube Gibson purified
Water 12 µL
Buffer 2.1 2 µL
Plasmid 5 µL
Enzyme EcoRI 0,5 µL
Enzyme PstI 0,5 µL


37°C, 60 min

PCR pDawn: whole fragment

Mix PCR preparation:
pDawn
MQ water 35,5 µL
Q5 Buffer 10 µL
dNTPs 10µM 1 µL
Primer Fwd 50µM 1µL pDawn Fwd I
Primer Rev 50µM 1µL pDawn Rev III-VI
DNA 1µL pDawn
Taq Pol 0.5 µL
PCR cycle – IGEM TAQ 4
T°C Time Cycle
Initial denaturation
98
30 sec
1
Denaturation
98
30 sec
17
Annealing
60
30 sec
Extension
72
3 min
Final extension
72
6 min
1
Hold
4
Infinite

Electrophoresis of PCR products

Expected results

Expected results

Results

Results

We get expected bands for our plasmid
No amplification of pDawn


22 July 15