Friday 23^rd September

Lab work

Visualization

NanoDrop Measurements

By Maxence, Manhaz & Coline

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