Tuesday 4^th October

Lab work

Visualization

Culture of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 and GFP 1.9 in pUC19 (pPS16_009) clone 1

By Maxence

As no clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) were obtained and as the concentrations of plasmids extracted the 2nd October were too low, the following clones were put on 2 x 10 mL liquid cultures (LB + 30 µg/mL Cm) grown at 37°C overnight:

• pPS16_018 (FKBP - GFP 10) clone 6
• pPS16_019 (FRB - GFP 11) clone 4

PCR of extracted GFP 1.9 in pSB1C3 (pPS16_009)

By Maxence

As no good clones were obtained for GFP 1.9 in pSB1C3 (pPS16_020) were obtained, a new PCR was run using extracted plasmids containing GFP 1.9 in pUC19 (pPS16_009) extracted the 2nd October. For each amplification, two matrix were used as two liquid cultures were set the 1st October.

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

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

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

Primers used were:

5 µL of PCR products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity.

PCR products expected were :

GEL

Cloning of GFP 1.9 PCR product in pSB1C3 by digestion-ligation

By Maxence

For that purpose, GFP 1.9 PCR product from today was cut by restriction enzymes PstI & XbaI as following:

For the most concentrated one (GFP 1.9 - 1):

• 10 µL of GFP 1.9 PCR product
• 4 µL of buffer FD
• 1 µL of restriction enzyme PstI
• 1 µL of restriction enzyme XbaI
• 24 µL of water

For the less concentrated one (GFP 1.9 - 2):

• 20 µL of GFP 1.9 PCR product
• 4 µL of buffer FD
• 1 µL of restriction enzyme PstI
• 1 µL of restriction enzyme XbaI
• 14 µL of water

Furthermore, BbaB0015 plasmid (in order to have pSB1C3) was cut by restriction enzymes PstI & XbaI and was treated by the alkaline phosphatase FastAP (dephosphorylation of cloning vector DNA to prevent recircularization during ligation) as following:

• 20 µL of BbaB0015 plasmid
• 4 µL of buffer FD
• 1 µL of restriction enzyme EcoRI
• 1 µL of restriction enzyme XbaI
• 1 µL of alkaline phosphatase FastAP
• 13 µL of water

The mix were incubated for 1 hour at 37°C. Then, 2 µL of each digestion products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

Migration products expected were :

GEL

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

Colony PCR of clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021)

By Maxence

For that purpose, X 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 :

GEL