Tuesday 20^th September

Lab work

Visualization

Samples preparation for sequencing

"By Maxence, Mahnaz & Coline"

20 µL of plasmids pSB1C3 FKBP - GFP 10 (clones 7, 8, 9 and 10) were sent to be sequenced. 20 µL of the primers iPS84 (5µM) and iPS140 (5µM) were sent for sequencing.

NanoDrop Measurements

"By Maxence, Mahnaz & Coline"

Colony PCR of the 4 clones containing GFP 1.9 in pSB1C3 (pPS16_020) from the 12th September

By Maxence, Mahnaz & Coline

As results from sequencing were not good, clones sent previously and stocked in glycerol (2, 7, 8 and 12) were put on plate the 19th September, as each colony may not not be homogenous enough. Another colony PCR was done and anothers primers were used.

For that purpose, the 4 clones were 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 :

GEL

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

By Maxence, Mahnaz & Coline

Gibson was performed with cleaned up PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 treated by DpnI, more precisely PCR product 1 obtained by iPS174 & iPS175 (plasmid) and PCR product 2 obtained by iPS173 & iPS176 (insert) with the following protocol:

For each 20μl of reaction, mix the following reagents :

• 0.63 µL of insert
• 1.2 µL of plasmid
• 8.17 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 correct dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) obtained by Gibson

By Maxence, Mahnaz & Coline

Dh5a cells were transformed with corrected pSB1C3 containing dCas9 ST - GFP 11 (pPS16_017), or controls (no buffer mix and plasmid alone) using the usual protocol.

Cloning of GFP 1.9 from pUC19 (pPS16_009) in pSB1C3 by digestion-ligation

By Maxence, Mahnaz & Coline

As difficulties were faced in order to obtain GFP 1.9 in pSB1C3 with Gibson strategy, the digestion-ligation strategy was preferred. For that purpose, GFP 1.9 PCR products from the 9th September (obtained with DMSO) were cut by restriction enzymes XbaI & SpeI. Before, 1/10 of the clean up PCR products were put on gel in order to assess the quantitiy of DNA left.

GEL

DNA quantity was enough to run digestion as following:

• 10 µL of GFP 1.9 PCR product from 9th September
• 2 µL of buffer FD
• 2 µL of restriction enzyme DpnI
• 2 µL of restriction enzyme SpeI
• 4 µL of water

Furthermore, pSB1C3 plasmid were cut by the same restriction enzymes as following:

• 1 µL of pSB1C3
• 2 µL of buffer
• 1 µL of restriction enzyme DpnI
• 1 µL of restriction enzyme SpeI
• 15 µL of water

The mix were incubated for 1 hour at 37°C. The digested products were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol. After clean-up, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

GEL

Once template and vector were cut, DNA ligase was used to join the sticky ends of the template and vector together. For that purpose, two protocols were used:

• 7 µL of template (GFP 1.9 PCR product from 9th treated by DpnI and SpeI)
• 1 µL of vector (pSB1C3 treated by DpnI and SpeI)
• 1 µL of Buffer T4 10X
• 1 µL of ligase T4 enzyme

And:

• 1 µL of template (GFP 1.9 PCR product from 9th treated by DpnI and SpeI)
• 1 µL of vector (pSB1C3 treated by DpnI and SpeI)
• 1 µL of Buffer T4 10X
• 1 µL of ligase T4 enzyme
• 6 µL of water

The mix were incubated for 1 hour at rooming temperature.

Two controls were done for further transformation application: one open plasmid and one ligated plasmid (without template).

Clean-up of GFP 1.9 in pSB1C3 (pPS16_020) obtained by Digestion-Ligation

By Maxence, Mahnaz & Coline

GFP 1.9 in pSB1C3 (pPS16_020) obtained by Digestion-Ligation was cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.

Transformation of DH5a cells with GFP 1.9 in pSB1C3 (pPS16_020) obtained by Digestion-Ligation

By Maxence, Mahnaz & Coline

Dh5a cells were transformed with pSB1C3 containing GFP 1.9 (pPS16_020), or controls (open plasmid and ligated plasmid) using the usual protocol.

Cloning of FRB - GFP 11 from pSB1C3 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation

By Maxence, Mahnaz & Coline

As difficulties were faced in order to obtain GFP 1.9 in pSB1C3 with Gibson strategy, we decided to construct first pPS16_021 (FRB - GFP 10 - FKBP - GFP 11 in pSB1C3) and then clone GFP 1.9 from pUC19 in it to construct pPS16_022 (FRB - GFP 10 - FKBP - GFP 11 - GFP 1.9 in pSB1C3). For that purpose, pPS16_019 was cut by restriction enzymes EcoRI & SpeI. Before, 1/10 of the extracted plasmid was put on gel in order to assess the quantitiy of DNA left.

GEL

DNA quantity was enough to run digestion as following:

• 10 µL of pPS16_019
• 2 µL of buffer
• 2 µL of restriction enzyme EcoRI
• 2 µL of restriction enzyme SpeI
• 4 µL of water

Furthermore, pPS16_018 was cut by restriction enzymes EcoRI & XbaI as following:

• 10 µL of pPS16_18
• 2 µL of buffer
• 1 µL of restriction enzyme EcoRI
• 2 µL of restriction enzyme XbaI
• 4 µL of water

The mix were incubated for 1 hour at 37°C. The digested products were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol. After clean-up, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

GEL

Once template and vector were cut, DNA ligase was used to join the sticky ends of the template and vector together. For that purpose, two protocols were used:

• 7 µL of template (pPS16_019 treated by EcoRI & SpeI)
• 1 µL of vector (pPS16_018 treated by EcoRI & XbaI)
• 1 µL of Buffer T4 10X
• 1 µL of ligase T4 enzyme

And:

• 1 µL of template (pPS16_019 treated by EcoRI & SpeI)
• 1 µL of vector (pPS16_018 treated by EcoRI & XbaI)
• 1 µL of Buffer T4 10X
• 1 µL of ligase T4 enzyme
• 6 µL of water

The mix were incubated for 1 hour at rooming temperature.

Two controls were done for further transformation application: one open plasmid and one ligated plasmid (without template).