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 Tuesday 6th September 1.1 Visualization 1.1.1 Q5 PCR on dCas9 ST - GFP 11 in pSB1C3, FRB in pJET and GFP 1.9 in pUC19 1.1.2 PCR Clean-up of PCR products 1.1.3 NanoDrop Measurements 1.1.4 Gel of cleaned up PCR products 1.1.5 Linearization of PCR product GFP 11 - pSB1C3 from clone 8 1.1.6 Transformation of DH5a cells with dCas9 NM - GFP 10 in PSB1C3 (pPS16_016) obtained by Gibson Tuesday 6th September Visualization Q5 PCR on dCas9 ST - GFP 11 in pSB1C3, FRB in pJET and GFP 1.9 in pUC19 By Maxence & Mahnaz Q5 PCR was performed on plasmids with the following protocol: For each 50μl of reaction, mix the following reagents : 1 µL of plasmid 1 µL of dNTPs (10mM) 1 µL of each primer mix (10µM) 10 µL of Q5 buffer (5X) 0,5 µL of Q5 high fidelity polymerase 35,5 µL of nuclease free water Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow: Step Temperature Time Initial denaturation 98°C 30sec 30 cycles 98°C 10sec Tm 20sec 72°C t Final Extension 72°C 2min Hold 4°C $\infty$ Primers used were: Matrix dCas9 ST - GFP 11 in pSB1C3 clones 6 and 8 FRB in pJET clones 4 and 9 GFP 1.9 in pUC19 Primers iPS152 and iPS151 iPS149 and iPS150 iPS84 and iPS140 Tm 57,5°C 56,7°C 72°C t 1 min 30 20 sec 30 sec PCR Clean-up of PCR products By Maxence & Mahnaz PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol. NanoDrop Measurements By Maxence & Mahnaz Sample Concentration (ng/µL) PCR fragment GFP 11 clone 6 187.23 PCR fragment GFP 11 clone 8 156.85 PCR fragment FRB clone 4 75.67 PCR fragment FRB clone 9 246.41 PCR fragment GFP 1.9 22.06 Gel of cleaned up PCR products By Maxence & Mahnaz After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. PCR products expected were : PCR products Expected band size (bp) GFP 11- pSB1C3 2500 FRB 374 GFP 1.9 862 Result of the migration No PCR products were obtained for GFP 1.9. Concerning FRB and GFP 11 - pSB1C3 amplification, good results were obtained and clones 9 for FRB and clone 8 for GFP 11 - pSB1C3 were selectionned. Linearization of PCR product GFP 11 - pSB1C3 from clone 8 By Maxence & Mahnaz PCR product GFP 11 - pSB1C3 has been linearized for further Gibson application by using DpnI treatment : 30 µL of cleaned up PCR product GFP 11 - pSB1C3 4 µL of fast digest buffer 1 µL of DpnI 5 µL of water The mix was placed 10 minutes at 37°C and was cleaned by using the NucleoSpin Gel and PCR Clean-up kit protocol. Transformation of DH5a cells with dCas9 NM - GFP 10 in PSB1C3 (pPS16_016) obtained by Gibson By Maxence & Mahnaz Dh5a cells were transformed with pSB1C3 containing dCas9 NM - GFP 10 (pPS16_016) (obtained by Gibson of fragment 1 PCR products (one with Q5 Pol and one with Phusion) + fragment 2 PCR products (one with Q5 Pol and one with Phusion) + pSB1C3), or containing control (no mix added when Gibson performed) using the usual protocol.
By Maxence & Mahnaz
Q5 PCR was performed on plasmids with the following protocol:
For each 50μl of reaction, mix the following reagents :
Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:
Primers used were:
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
PCR products expected were :
No PCR products were obtained for GFP 1.9. Concerning FRB and GFP 11 - pSB1C3 amplification, good results were obtained and clones 9 for FRB and clone 8 for GFP 11 - pSB1C3 were selectionned.
PCR product GFP 11 - pSB1C3 has been linearized for further Gibson application by using DpnI treatment :
The mix was placed 10 minutes at 37°C and was cleaned by using the NucleoSpin Gel and PCR Clean-up kit protocol.
Dh5a cells were transformed with pSB1C3 containing dCas9 NM - GFP 10 (pPS16_016) (obtained by Gibson of fragment 1 PCR products (one with Q5 Pol and one with Phusion) + fragment 2 PCR products (one with Q5 Pol and one with Phusion) + pSB1C3), or containing control (no mix added when Gibson performed) using the usual protocol.
