Toggle navigation Home Team Team Media Collaborations Sponsors Acknowledgements Project Background Design CRISPR/Cas9 Strategy Experiments Notebook Results Perspective Interlab Study Parts Parts Basic Parts Composite Parts Human Pratices Overview Societal Issues of CRISPR/Cas9 Responsible Research and Innovation GMO regulation Integrated Practices Engagement Model Attributions Safety Contents 1 Thursday 7th July 1.1 Lab work 1.1.1 Bringing DNA closer 1.1.1.1 CAS9 q5 high fidelity PCR 1.1.1.2 PZA21 q5 high fidelity PCR 1.1.2 Biobrick characterization 1.1.2.1 Digestion of the plasmid psB1C3 containing the biobrick K1327001 1.1.3 Visualization 1.1.3.1 DH5a|pUC19_gBlock Stock of glycerol 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 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. 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%)
By Caroline and Léa
1L of agarose gel TAE 0,5X agarose 0,8% was prepared following the usual protocol
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.
‘’By Naiane’’
PZA21 plasmids were amplified with primers:
Primers stocks (100µM) were diluted as follows:
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.
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.
Then, plasmidic DNA of the remaining Clone 1 and 2 (from the extraction of 06/07/16) were re-concentrated using the following protocol:
By Mathilde and Laetitia
Transformed cells containing gBlock:
For each sample, we made a stock puting 500 of culture and 250 of glycérol (60%)
