Team:GO Paris-Saclay/labnotebook-cpg2-folc
Cpg2-FolC experiments
9/08, by Britany
- Digestion of pPS18-13.5 by SpeI and PstI
10 µL | pPS18-13.5 DNA | 375 ng | |
1 µL | PstI | ||
1 µL | SpeI | ||
2 µL | Buffer CutSmart | ||
6 µL | Water | ||
Final volume | 20 µL | ||
DNA was digested 1h at 37°C.
- Electrophoresis gel of digestion and gel clean-up :
Migration step lasted 20 minutes at 100V with a 0,8% agarose gel.
Spin column gel cleanup was done according to the protocol [NucleoSpin Macherey-Nagel].
Expected size | Obtained size | |||
pPS18-13.5 | 3,4 kpb | pPS18-13.5 digested | 3,5 kpb 2,2 kpb | |
This new size obtained can be our vector without the insert cpg2. So, we purified our DNA and we obtained our expected size.
10/08, by Arthur and Julie M
- Digestion of pPS18-17.2, pPS18-17.5 and pPS18-17.7 by XbaI and PstI
12 µL 13 µL 10 µL | pPS18-17.2 DNA pPS18-17.5 DNA pPS18-17.7 DNA |
1 µL | PstI |
1 µL | XbaI |
2 µL | Buffer CutSmart |
Qsp | Water |
Final volume | 20 µL |
DNA was digested 1h at 37°C.
- Inactivation of XbaI and PstI 20 min at 80°C.
- Digestion by SmaI to cut the vector unneeded. DNA was digested 1h at 37°C
- Ligation to achieve pPS18-20 (pSB1C3/cpg2/folC)
2 µL of vector pPS18-13.5 purified and digested (18 ng) were ligate with 6 µL of each insert folC (from pPS18-17.2, pPS18-17.5 or pPS18-17.7) (11 ng). Ligation was let overnight in the fridge.
13/08, by Britany
- Transformation of the ligation product (pPS18-20)
DH5α competent cells were transformed with 4 µL of our ligation products (10/08). After heat choc, LB was added and cells were left to grow 2h at 37°C. Cell cultures were spread on two LB + Cm (30 ng/µL) plates (1/10 of culture then the rest).
14/08, by Britany
- Trasformation results : we had some clones on each plates (around 20) and we had nothing on negative control.
- Colony PCR
12 clones by each contruction were tested with this PCR.
Protocol | Programme | ||||
DNA + Water | 22,4 µL each several tubes | Cycle step | Protocol | Cycles | |
Temperature | Time | ||||
2.5 mM dNTPs | 2 µL | Initial Denaturation | 95°C | 3 min | 1 |
Primer A (VF2) 10 µM | 1,25 µL | Denaturation | 95°C | 30s | 30 |
Primer B (VR) 10 µM | 1,25 µL | Annealing | 49°C | 30s | |
DreamTaq polymerase | 0,1 µL | Extension | 72°C | 3 min 30s | |
10X GreenTaq buffer | 2,5 µL | Final Extension | 72°C | 5 min | 1 |
Final volume | 30 µL | 16°C | hold | ||
20/08, by Britany
- Electrophoresis gel of colony PCR :
Migration step lasted 30 minutes at 100V with a 0,8% agarose gel.
Expected size | Obtained size | |||
pPS18-20 | 3,4 kpb | pPS18-20.6 | Good size pPS18-20.6.5 | |
pPS18-20.10 | Good size pPS18-20.10.1, pPS18-20.10.4 and pPS18-20.10.6 | |||
pPS18-20.8 | Good size pPS18-20.8.3 and pPS18-20.8.7 | |||
- Culture of 6 clones in 5 mL of LB + Cm.
21/08, by Britany
- DNA extraction of our 6 cultures
Sample | Concentration | [protein] / [DNA] |
pPS18-20.6.5 | 190,3 ng/µL | 1,87 |
pPS18-20.10.1 | 155,1 ng/µL | 1,87 |
pPS18-20.10.4 | 170,0 ng/µL | 1,86 |
pPS18-20.10.6 | 161,0 ng/µL | 1,85 |
pPS18-20.8.3 | 143,5 ng/µL | 1.87 |
pPS18-20.8.7 | 162,1 ng/µL | 1.87 |
- Digestion of each pPS18-20 by ApaLI
0,5 µL | pPS18-20 DNA |
1 µL | ApaLI |
1 µL | Buffer Tango 10X |
7,5 µL | Water |
Final volume | 10 µL |
DNA was digested 1h30 at 37°C.
- Electrophoresis gel of digestion :
Migration step lasted 20 minutes at 100V with a 0,8% agarose gel.
Expected size | Obtained size | |||
pPS18-20 | 1260 pb 3960 Pb | For all clones | 1200 pb 4 kpb | |
According to these results, our 6 pPS18-20 clones are good ones.