Friday 23rd September
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
|
As gblock 2.2 and FKBP were too concentrated, they were diluated at 1/10.
Gibson of cleaned up PCR products FKBP from 8th September x gblock 2.2 from the 8th September x pSB1C3 treated by DpnI and Gibson of PCR product containing FKBP - GFP 10 from the 22nd September x pSB1C3 treated by DpnI
By Maxence, Manhaz & Coline
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 :
- 1.74 µL of insert 1
- 1.78 µL of insert 2
- 1.67 µL of plasmid
- 4.81 µL of water
- 10 µL of buffer mix
Furthermore, Gibson was performed with cleaned up PCR containing FKBP - GFP 10 (insert) x pSB1C3 treated by DpnI (plasmid) with the following protocol:
For each 20μl of reaction, mix the following reagents :
- 1.89 µL of insert
- 1.67 µL of plasmid
- 6.44 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 2, 7 and 8 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 2
- pPS16_017 (dCas9 ST - GFP 11) clone 7
- pPS16_017 (dCas9 ST - GFP 11) clone 8
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
Plasmids extraction of clones 2, 7 and 8 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 2
- pPS16_017 (dCas9 ST - GFP 11) clone 7
- pPS16_017 (dCas9 ST - GFP 11) clone 8
Glycerol stocks of clones 3, 4, 7 and 8 containing GFP 1.9 in pSB1C3 (pPS16_020)
By Maxence, Mahnaz & Coline
The glycerol stock of the bacteria with the following plasmids were made.
- pPS16_020 (GFP 1.9) clone 3
- pPS16_020 (GFP 1.9) clone 4
- pPS16_020 (GFP 1.9) clone 7
- pPS16_020 (GFP 1.9) clone 8
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
Plasmids extraction of clones 3, 4, 7 and 8 containing GFP 1.9 in pSB1C3 (pPS16_020)
By Maxence, Mahnaz & Coline
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
- pPS16_020 (GFP 1.9) clone 3
- pPS16_020 (GFP 1.9) clone 4
- pPS16_020 (GFP 1.9) clone 7
- pPS16_020 (GFP 1.9) clone 8
