Thursday 7th 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 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 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
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%)
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 37°c
