Thursday 7^th July

Lab work

By Caroline and Léa

1L of agarose gel TAE 0,5X agarose 0,8% was prepared following the usual protocol

Bringing DNA closer

CAS9 q5 high fidelity PCR

By Léa

Primers IPS 134 and IPS 135 were used to amplify TD-CAS9, NM-cas9 and ST1-cas9. Primers SP1 (IPS 134 + IPS 136) and SP2 (IPS 137 + IPS 135) were used for DS-SPCasN- . Primers were diluted in order to get a 100µM final concentration. The usual protocol for polymerase q5 PCR was followed, with a Tm=65°c.

PZA21 q5 high fidelity PCR

‘’By Naiane’’

PZA21 plasmids were amplified with primers:

• Ter_SPdcas_R and Link-SPdcas_R
• Link-TDdcas_F and Ter_TDdcas_R
• Ptet R and Ptet F

Primers stocks (100µM) were diluted as follows:

• Add 280µL of H2O nuclease free to Ter_SPdcas_R and 558 µL to Link-SPdcas_R
• Add 599µL of H2O nuclease free to Link-TDdcas_F and 219µL to Ter_TDdcas_R
• Add 191µL of H2O nuclease free to Ptet F and 315µL to Ptet R.

Migration of NM, TD, ST1, ST2, SP, TD, pTet

Biobrick characterization

Pre-culture of BL21

By Laetitia and Charlène

One colony from BL21 was deposited in 4mL of LB and put in incubation at 37°c, 200 rpm ON.

Digestion of the plasmid psB1C3 containing the biobrick K1327001

By Mathilde and Alice 

For each sample: 10 μL of DNA 2 μL fast digest buffer 1 μL Pst 1 6 μL H2O

Samples were incubated 5 min at 37°C. Loading Buffer was added for each solution. Samples were put on an agarose gel for an electrophoresis.

Migration of Cl1(K13) and Cl2 (K13)

Clone 1 didn't show any bands. Clone 2 showed bands at 2000 kb (corresponding to the size of psB1C3) and 1500 kb (corresponding to the size of biobrick K1327001)

We concluded that clone 2 contain the right insert.

Visualization

Electrophoresis migration for gBlocks PCR products

By Caroline and Léa

PCR products of pPS16_001, pPS16_002, pPS16_003 pPS16_004, pPS16_005 and pPS16_006 from the Team:Paris_Saclay/Notebook/July/6#Visualization│day before were each added to 4µL of violet gel loading dye. No amplicon was detected.

Results of DH5α|pPS16_004, pPS16_004 clone 6 and pPS16_007 cultures

Cultures from the Team:Paris_Saclay/Notebook/July/6#Visualization│day before showed absolutely no blue colony. We suspect a problem with X-Gal IPTG.

DH5α|pPS16_004, pPS16_004 clone 6 and pPS16_007 cultures

‘’ By Laetitia and Charlène’’ In order to solve the X-Gal IPTG issue we had with the previous transformed gBlocks cultures, and to test X-Gal IPTG efficiency, four different mediums were used :

• New X-Gal, Old X-Gal
• Old X-Gal, New IPTG
• New X-Gal, New IPTG
• Old X-Gal, Old IPTG

A blue colony was plated on each of those four mediums LB + Ampicillin (50oµL) + X-Gal IPTG (1/1000). 100 µL of DH5α|pPS16_004, pPS16_004 clone 6 and pPS16_007 were plated again on LB + Ampicillin (50µg/mL) + X-Gal IPTG

Then, plasmidic DNA of the remaining Clone 1 and 2 (from the extraction of 06/07/16) were re-concentrated using the following protocol:

• Add 2 volumes of cold ethanol (100%)
• Add the quivalent of1/10 of the remaining DNA of solution III
• Put it 30 min at -20°C
• Centrifuge 4 min at1700 rpm
• Remove the supernatant
• Add 1 mL of ethanol at 70% and inverse
• Centrifuge 4 min at 1300 rpm
• Let it dry 1 h
• Dilute with 10 L of sterilized water.
• Put it at -20°C

Visualization

DH5a|pUC19_gBlock Stock of glycerol

By Mathilde and Laetitia

Transformed cells containing gBlock:

• 1.1 (6clones)
• 1.2 (6clones)
• 2.1(6clones)
• 2.2(6clones)
• 3.1(6clones)
• 3.2(6clones)

For each sample, we made a stock puting 500 of culture and 250 of glycérol (60%)