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
Cas9 PCR
By Léa
Forward primer Sequencing addgene Cas plasmids (IPS 134) and Reverse primer Sequencing addgene Cas plasmids (IPS 135) were used for NM Cas9(DS-NMcas), ST1 Cas9 (DS-ST1casN-) and Td Cas9 (DS-TDcasN-).
For SP Cas9 (DS-SPcasN-) we used primers :
- IPS 134 + IPS 136 = SP1
- IPS 137 + IPS 135 = SP2
The PCR was conducted following the usual protocol
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.
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
PCR products migration
By Caroline and Léa
gBlocks screened the 06/07/2016 were put on migration.
The usual protocol was followed.
20µL of each PCR product was added to 4µL of purple loading dye 6X.
TODO PHOTO GEL pas dans le cahier !
Cultures results
There was no blue colony observed on xGal/IPTG + ampicillin mediums for transformed transformed pPS16_004, pPS16_007 and the pPS16_007 clone 6 gBlocks.
Culture of transformed pPS16_004, pPS16_007 and the pPS16_007 clone 6
By Laetitia and Charlène
Because of the absence of blue colony after culture, xGal and IPTG efficency was tested.
A petri dishe was splitted in four parts :
- new xGal, new IPTG
- new xGal, former IPTG
- former xGal, new IPTG
- former xGal, former IPTG
A blue colony from the control tube 2 of the 04/07/2016 was plated on each of the four part on medium LB + Ampicillin (100µg/mL) + xGal/IPTG (1/1000).
100µ of pPS16_004, pPS16_007 or the pPS16_007 clone 6 bacteries were plated again on LB + Ampicillin (100µg/mL) + xGal/IPTG (1/1000) medium.
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 putting 500 of culture and 250 of glycerol (60%).
Then, the remaining plasmid DNA from Clone 1 and 2 (from the 06/07/16 extraction) were re-concentrated using the following protocol:
- Add 2 volumes of cold ethanol (100%)
- Add the equivalent of 1/10 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
