Monday 22th August
Visualization
Dream Taq PCR of GFP1-9 cloned in the pSB1C3
By Mathilde
Clones 7,8,9 and 10 of GFP1-9 cloned in the pSB1C3 were amplified following the protocol :
- 1,5µL of DreamTaq Buffer
- 1µL of dNTPs
- 1µL of each primers IPS83 and IPS84 (10 µM)
- 0,13µL of Dream Taq Polymerase
- 19,37 µL of H20
Colonies were put into sterile water, with 5 minutes of denaturation (95°C in the PCR machine) before the addition of the mix.
Annealing temperature was 57°c.
After amplification, 1µL of each PCR product and 10µL of the DNA purple ladder were loaded into a 0,8% agarose gel + BET and then migrated at 100V for 30min.
Sequencing of 1.2, FKBP, FRB cloned in pJET plasmid
Terrence and Léa
Was sent :
|
|
Clone
|
Concentration
|
size
|
| 1.2
|
11
|
392.66
|
3934
|
| FKBP
|
3
|
272.37
|
3393
|
| FKBP
|
4
|
404.65
|
3393
|
| FKBP
|
5
|
204.87
|
3393
|
| FRB
|
2
|
256.44
|
3347
|
| FRB
|
5
|
129.86
|
3347
|
Purification on gel of gBlocks 3 and 4
Terrence and Mathilde
After amplification, 50µL of each PCR product diluted 6 times with 10 µL of the gel loading dye, and 10µL of the DNA purple ladder were put into a 0,8 agarose gel + BET and migrated at 100V for 30min.
The extraction was carried out with the usual protocol.
Nano Drop :
|
|
size (ng/µL)
|
260/230
|
260/280
|
| Fragment 3
|
11.07
|
0.48
|
3.02
|
| Fragment 4
|
50.12
|
1.07
|
1.93
|
Purification of PSB1C3 with kit
Terrence
pSB1C3 was purified following the usual protocol.
To remove any circular plasmid pSB1C3, it was digested with the enzyme DPNI.
Nano Drop :
|
|
size (ng/µL)
|
260/230
|
260/280
|
| PSB1C3
|
58.66
|
0.74
|
1.71
|
Gibson on fragment 3 and fragment 4
Terrence
We have assembled the fragment 3 and 4 following the HiFi assembly protocolwith 50ng of vector and 2 times more of insert.
Here is the volume used for the Gibson :
| Components
|
Volume (µL)
|
| PSB1C2
|
0.85
|
| Fragment 3
|
6.83
|
| Fragment 4
|
1.60
|
| H2O
|
0.72
|
We made 2 tubes composed of this upper mix.
In the control one, we added 10 µL of water, whereas in the "Gibson" one, we added 10µL of Gibson mix.
Gibson assembly product (fragments 3 and 4 ligation) and control were transformed into DH5a cells, using 2 µL of DNA, and following the usual protocol.
GFP1-9 and psB1C3 ligation product transformation in DH5a
By Léa
The GFP1-9 and pSB1C3 ligation products of the 10/08/2016 was transformed into DH5a cells, using 2 µL of DNA, and following the usual protocol.
Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C o/n.
Interlab Study
Transformation into DH5a cells
By Léa
Devices 2 and 3, negative control and positive control were transformed into DH5a cells, following the usual protocol.
Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C o/n.
Device 1 culture
By Léa
Device 1 transformed DH5a glycerol stock was plated on LB Agar chloramphenicol Petri Dishes and incubated at 37°C o/n.
Cell culture
By Mahnaz
Bacteria containing plasmids coding NM Cas9 (DS-NMcas)which have been ordered from Addgene were put into 3mL of LB medium containing 50µg/mL of spectinomycin and incubated overnight at 37°C, 180 rpm.
