Monday 19^th September

Visualization

Glycerol stocks of clones 7, 8, 9 and 10 containing FKBP - GFP 10 in pSB1C3 (pPS16_018)

"By Maxence, Mahnaz, Coline & Caroline"

The glycerol stock of the bacteria with the following plasmids were made.

• pPS16_018 (FKBP - GFP 10) clone 7
• pPS16_018 (FKBP - GFP 10) clone 8
• pPS16_018 (FKBP - GFP 10) clone 9
• pPS16_018 (FKBP - GFP 10) clone 10

500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.

Plasmids extraction of clones 7, 8, 9 and 10 containing FKBP - GFP 10 in pSB1C3 (pPS16_018)

"By Maxence, Mahnaz Coline & Caroline"

The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:

• pPS16_018 (FKBP - GFP 10) clone 7
• pPS16_018 (FKBP - GFP 10) clone 8
• pPS16_018 (FKBP - GFP 10) clone 9
• pPS16_018 (FKBP - GFP 10) clone 10

Colony PCR of 8 clones containing GFP 1.9 in pSB1C3 (pPS16_020) from the 12th September

By Maxence, Mahnaz, Coline & Caroline

Seqencing of clones 2, 7, 8 and 12 from the 14th September was not good as empty plasmids were obtained. Another colony PCR was done for 8 clones from the 12th September but anothers primers were used.

For that purpose, 8 clones were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.

For each clones contained in 20 μl water, 5.13 μL of the following mix were added :

• 2.5 µL DreamTaq Buffer
• 0.5 µL of dNTPs (10mM)
• 1 µL of each primer mix (10µM)
• 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Primers used were:

After amplification, 3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

Furthermore, plasmid extacted from clones containing GFP 1.9 in pSB1C3 (pPS16_018) were also put on the gel.

Result of the migration

Plasmids extracted from clones containing GFP 1.9 in pSB1C3 (pPS16_018) were good, but no PCR products were obtained for GFP 1.9, cloning failed.

PCR of pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 to correct mutations

By Maxence, Mahnaz, Coline & Caroline

In order to correct mutations in dCas9 ST - GFP 11, PCR was performed with the following protocol.

For each 50μl of reaction, mix the following reagents :

• 1 µL of matrix
• 1 µL of dNTPs (10mM)
• 2.5 µL of each primer mix (10µM)
• 10 µL of buffer HF (5X)
• 0,5 µL of Phusion polymerase
• 32.5 µL of nuclease free water

Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:

Primers used were:

Gel of PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8

By Maxence, Mahnaz, Coline & Caroline

4 µL of each PCR products and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

Result of the migration

PCR products were obtained at the good size.

PCR Clean-up of PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8

By Maxence, Mahnaz, Coline & Caroline

PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.

Linearization of cleaned-up PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8

By Maxence, Mahnaz, Coline & Caroline

Cleaned-up PCR product has been linearized for further Gibson application by using DpnI treatment :

• 30 µL of cleaned up PCR product
• 4 µL of fast digest buffer
• 1 µL of DpnI
• 5 µL of water

The mix was placed 10 minutes at 37°C and was cleaned by using the NucleoSpin Gel and PCR Clean-up kit protocol.

Clean-up of PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 treated by DpnI

By Maxence, Mahnaz, Coline & Caroline

PCR products treated by DpnI were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.