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
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
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
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 |
T°C | Time | Cycle | |
---|---|---|---|
Initial denaturation | |||
Denaturation | |||
Annealing | |||
Extension | |||
Final extension | |||
Hold |
Expected results
Results
There were some amplifications of DNA materials but which one ????
PCR pDawn III
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 |
T°C | Time | Cycle | |
---|---|---|---|
Initial denaturation | |||
Denaturation | |||
Annealing | |||
Extension | |||
Final extension | |||
Hold |
Digestion of pDawn (PCR and Plasmid)
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
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