Monday 22^th August

Visualization

Dream Taq PCR of GFP1-9 cloned in the pSB1C3

By Mathilde

Clones 7,8,9 and 10 of GFP1-9 cloned in the pSB1C3 were amplified following the protocol :

• 1,5µL of DreamTaq Buffer
• 1µL of dNTPs
• 1µL of each primers IPS83 and IPS84 (10 µM)
• 0,13µL of Dream Taq Polymerase
• 19,37 µL of H20

Colonies were put into sterile water, with 5 minutes of denaturation (95°C in the PCR machine) before the addition of the mix. Annealing temperature was 57°c. After amplification, 1µL of each PCR product and 10µL of the DNA purple ladder were loaded into a 0,8% agarose gel + BET and then migrated at 100V for 30min.

Result of the PCR

Sequencing of 1.2, FKBP, FRB cloned in pJET plasmid

Terrence and Léa

Was sent :

Purification on gel of gBlocks 3 and 4

Terrence and Mathilde

After amplification, 50µL of each PCR product diluted 6 times with 10 µL of the gel loading dye, and 10µL of the DNA purple ladder were put into a 0,8 agarose gel + BET and migrated at 100V for 30min. The extraction was carried out with the usual protocol.

Nano Drop :

Gel for the extraction

Purification of PSB1C3 with kit

Terrence

pSB1C3 was purified following the usual protocol. To remove any circular plasmid pSB1C3, it was digested with the enzyme DPNI.

Nano Drop :

Gibson on fragment 3 and fragment 4

Terrence

We have assembled the fragment 3 and 4 following the HiFi assembly protocolwith 50ng of vector and 2 times more of insert. Here is the volume used for the Gibson :

We made 2 tubes composed of this upper mix. In the control one, we added 10 µL of water, whereas in the "Gibson" one, we added 10µL of Gibson mix.

Gibson assembly product (fragments 3 and 4 ligation) and control were transformed into DH5a cells, using 2 µL of DNA, and following the usual protocol.

GFP1-9 and psB1C3 ligation product transformation in DH5a

By Léa

The GFP1-9 and pSB1C3 ligation products of the 10/08/2016 was transformed into DH5a cells, using 2 µL of DNA, and following the usual protocol. Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C o/n.

Interlab Study

Transformation into DH5a cells

By Léa

Devices 2 and 3, negative control and positive control were transformed into DH5a cells, following the usual protocol. Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C o/n.

Device 1 culture

By Léa

Device 1 transformed DH5a glycerol stock was plated on LB Agar chloramphenicol Petri Dishes and incubated at 37°C o/n.

Cell culture

By Mahnaz

Bacteria containing plasmids coding NM Cas9 (DS-NMcas)which have been ordered from Addgene were put into 3mL of LB medium containing 50µg/mL of spectinomycin and incubated overnight at 37°C, 180 rpm.