Template:IONIS Paris/Notebook/29-07-15

Gel extraction of the 3 bands of pDawn

Miniprep of liquid culture with the red colonies

We used a kit from QIAgen

Digestion of pDawn and pSB1C3

Mix digestion preparation:
Tube pDawn Miniprep
Water 12 µL 12 µL
Buffer 2.1 2 µL 2 µL
Plasmid 5 µL 5 µL
Enzyme EcoRI 0,5 µL 0,5 µL
Enzyme SpeI 0,5 µL 0,5 µL

37°C, 60 min

Expected results

Expected results

Results

Results

We get 2 bands after the digestion of pDawn meaning a non-expected restriction site appeared into the sequence during the PCR useless to answer biobrick standard requirement.
The profile of the Gibson digestion correspond to pSB1C3 with the RFP (totally unexpected since we have purifies the digested backbone).

PCR pDawn III

Mix PCR preparation:
pDawn III
MQ water 26,5 µL
Q5 Buffer 10 µL
High GC content Buffer 10 µL
dNTPs 10µM 1 µL
Primer Fwd 50µM 0,5 µL pDawn Fwd VI
Primer Rev 50µM 0,5 µL pDawn Rev III-VI
DNA 1µL pDawn III PCR1
Taq Pol 0.5 µL
PCR cycle – IGEM TAQ
T°C Time Cycle
Initial denaturation
98
5 min
1
Denaturation
98
30 sec
17
Annealing
60
30 sec
Extension
72
2 min
Final extension
72
10 min
1
Hold
4
Infinite
Expected results

Expected results

Results

Results

There were some amplifications of DNA materials but which one ????

PCR pDawn III

Mix PCR preparation:
pDawn III
MQ water 40 µL
RB Buffer 5 µL
Mg2+ 1,5 µL
dNTPs 10µM 1 µL
Primer Fwd 50µM 0,5 µL pDawn Fwd VI
Primer Rev 50µM 0,5 µL pDawn Rev III-VI
DNA 1µL pDawn III PCR1
Taq Pol 0.5 µL
PCR cycle – IGEM TAQ
T°C Time Cycle
Initial denaturation
98
5 min
1
Denaturation
98
30 sec
17
Annealing
59-60-61
30 sec
Extension
72
2 min
Final extension
72
10 min
1
Hold
4
Infinite

Digestion of pDawn (PCR and Plasmid)

Mix digestion preparation:
Tube pDawn PCR pDawn PCR pDawn PCR pDawn pDawn
Water 12 µL 12 µL 12 µL 12 µL 12 µL
Buffer 2.1 2 µL 2 µL 2 µL 2 µL 2 µL
Plasmid 5 µL 5 µL 5 µL 5 µL 5 µL
Enzyme EcoRI 0,5 µL / / 0,5 µL /
Enzyme SpeI / 0,5 µL / / 0,5 µL
Enzyme XbaI / / 0,5 µL / /

37°C, 60 min

Expected results

Expected results

Results

Results

Comparison of digestion profile between the original plasmid and the fragment amplificated by PCR. Confirmation of a new restriction site into pDawn PCR


29 July 15