Contents
- 1 Friday 23rd September
- 1.1 Lab work
- 1.1.1 Visualization
- 1.1.1.1 NanoDrop Measurements
- 1.1.1.2 Gibson of cleaned up PCR products FKBP from 8th September x gblock 2.2 from the 8th September x pSB1C3 treated by DpnI
- 1.1.1.3 Transformation of DH5a cells with FKBP - GFP 10 in pSB1C3 (pPS16_018) obtained by Gibson
- 1.1.1.4 Glycerol stocks of clones X, X, X and X containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
- 1.1.1.5 Plasmids extraction of clones X, X, X and X containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
- 1.1.1.6 Digestion of plasmids extracted from clones X, X, X and X containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
- 1.1.1.7 Glycerol stocks of clones X, X, X and X containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)
- 1.1.1.8 Plasmids extraction of clones X, X, X and X containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)
Friday 23rd September
Lab work
Visualization
NanoDrop Measurements
By Maxence, Manhaz & Coline
| Sample
|
Concentration (ng/µL)
|
| FKBP amplification product from the 8th September
|
231.55
|
| gblock 2.2 amplification product from the 8th September
|
183.9
|
| pSB1C3 treated by DpnI from the 15th September
|
59.68
|
| PCR product containing FKBP - GFP 10 from the 22nd September
|
38.19
|
Gibson of cleaned up PCR products FKBP from 8th September x gblock 2.2 from the 8th September x pSB1C3 treated by DpnI
By Maxence
Gibson was performed with cleaned up PCR products FKBP (insert 1) (obtained the 8th September) x gblock 2.2 (insert 2) (obtained the 8th September) x pSB1C3 treated by DpnI (plasmid) with the following protocol:
For each 20μl of reaction, mix the following reagents :
- 0.31 µL of insert 1
- 0.2 µL of insert 2
- 2 µL of plasmid
- 7.49 µL of water
- 10 µL of buffer mix
Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). The PCR was performed as follow : 1 hour at 50°C.
Transformation of DH5a cells with FKBP - GFP 10 in pSB1C3 (pPS16_018) obtained by Gibson
By Maxence & Mahnaz
Dh5a cells were transformed with pSB1C3 containing FKBP - GFP 10 (pPS16_018), or controls (no buffer mix and plasmid alone) using the usual protocol.
Glycerol stocks of clones X, X, X and X containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
"By Maxence, Mahnaz & Coline"
The glycerol stock of the bacteria with the following plasmids were made.
- pPS16_022 (GFP 1.9) clone X
- pPS16_022 (GFP 1.9) clone X
- pPS16_022 (GFP 1.9) clone X
- pPS16_022 (GFP 1.9) clone X
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
Plasmids extraction of clones X, X, X and X containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
"By Maxence, Mahnaz & Coline"
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
- pPS16_022 (GFP 1.9) clone X
- pPS16_022 (GFP 1.9) clone X
- pPS16_022 (GFP 1.9) clone X
- pPS16_022 (GFP 1.9) clone X
Digestion of plasmids extracted from clones X, X, X and X containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
"By Maxence, Mahnaz & Coline"
To assess if DH5a cells were transformed with the good plasmid, plasmid digestion was run with XbaI as following:
- 1 µL of extracted plasmid
- 1 µL of buffer
- 1 µL of restriction enzyme XbaI
- 7 µL of water
A control was done with 1 µL of non-digested plasmid.
GEL
Glycerol stocks of clones X, X, X and X containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)
"By Maxence, Mahnaz & Coline"
The glycerol stock of the bacteria with the following plasmids were made.
- pPS16_017 (dCas9 ST - GFP 11) clone X
- pPS16_017 (dCas9 ST - GFP 11) clone X
- pPS16_017 (dCas9 ST - GFP 11) clone X
- pPS16_017 (dCas9 ST - GFP 11) clone X
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
Plasmids extraction of clones X, X, X and X containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)
"By Maxence, Mahnaz & Coline"
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
- pPS16_017 (dCas9 ST - GFP 11) clone X
- pPS16_017 (dCas9 ST - GFP 11) clone X
- pPS16_017 (dCas9 ST - GFP 11) clone X
- pPS16_017 (dCas9 ST - GFP 11) clone X
