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 Wednesday 5th October 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 1.1.1.2 Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation Wednesday 5th October Lab work Visualization Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 "By Maxence & Yacine" The following plasmids were extracted using a "standard Plasmid Miniprep" kit: pPS16_018 (FKBP - GFP 10) clone 6 pPS16_019 (FRB - GFP 11) clone 4 Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation By Maxence & Yacine For that purpose, FRB - GFP 11 (pPS16_019) clone 4 was cut by restriction enzymes XbaI & PstI as following: 20 µL of FRB - GFP 11 (pPS16_019) clone 4 4 µL of buffer FD 2 µL of restriction enzyme XbaI 2 µL of restriction enzyme PstI 10 µL of water And FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6 was cut by restriction enzymes PstI & XbaI and was treated by the alkaline phosphatase FastAP (dephosphorylation of cloning vector DNA to prevent recircularization during ligation) as following: 20 µL of FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6 4 µL of buffer FD 2 µL of restriction enzyme SpeI 2 µL of restriction enzyme PstI 2 µL of alkaline phosphatase FastAP 10 µL of water The mix were incubated for 20 minutes at 37°C. Then, 40 µL of each digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. Migration products expected were : Migration products Expected band size (bp) Template digested (pPS16_019 treated by XbaI & PstI) 727 Vector digested (pPS16_018 treated by SpeI & PstI) 2784 GEL The digestion products were cleaned up from the gel by using the NucleoSpin Gel and PCR Clean-up kit protocol.
"By Maxence & Yacine"
The following plasmids were extracted using a "standard Plasmid Miniprep" kit:
By Maxence & Yacine
For that purpose, FRB - GFP 11 (pPS16_019) clone 4 was cut by restriction enzymes XbaI & PstI as following:
And FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6 was cut by restriction enzymes PstI & XbaI and was treated by the alkaline phosphatase FastAP (dephosphorylation of cloning vector DNA to prevent recircularization during ligation) as following:
The mix were incubated for 20 minutes at 37°C. Then, 40 µL of each digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
Migration products expected were :
GEL
The digestion products were cleaned up from the gel by using the NucleoSpin Gel and PCR Clean-up kit protocol.
