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 11th August 1.1 Visualization 1.1.1 Transformation of DH5a cells with pPS16_020 1.1.2 pPS16_010 extraction 1.1.3 Extraction of pPS16_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 11) and pPS16_009(clone 7) 1.1.4 High Fidelity Phusion PCR of transformed cell with ligation 2 and 3 1.1.5 DreamTaq PCR of transformed cell with pPS16_002,008,010, 011, 012, 013, 014 and puC19 1.1.6 Phusion PCR on PSB1C3 1.1.7 Result of the ligation 2-3 PCR. 1.2 Bringing DNA closer 1.2.1 Extraction of DS-SPcasN- and DS-TDcasN- Thursday 11th August Visualization Transformation of DH5a cells with pPS16_020 By Léa Dh5a cells were transformed with pPS16_020 (GFP 1-9 ligated with pSB1C3 digested), or the linearized pSB1C3 (control), or not transformed (control) using the usual protocol. pPS16_010 extraction By Alice After colony screening PCR, 3 clones (clones 3, 7 and 8) seemed to have the insert of interest. Plasmids from these clones were extracted following this protocol. After extraction, plasmids were migrated on a gel. Band size expected: Plasmid name Plasmid size (bp) pPS16_010 3070 Migration of FRB plasmid Nano Drop: Plasmid name Concentration (ng/µL) 260/230 260/280 pPS16_010 clone 3 334.48 2.35 1.94 pPS16_010 clone 7 321.06 2.32 1.98 pPS16_010 clone 8 139.13 2.10 1.88 Extraction of pPS16_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 11) and pPS16_009(clone 7) By Terrence The extraction was carried out following the usual protocol. Extraction of pPS16_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 3 and 11) and pPS16_009 (clone 7) Nano Drop: Plasmid name Concentration (ng/µL) 260/230 260/280 pPS16_011 clone 3 34.08 1.54 1.57 pPS16_011 clone 4 368.42 2.36 1.92 pPS16_011 clone 6 295.99 2.29 1.91 pPS16_014 clone 11 529.17 2.39 1.91 pPS16_002 clone 7 30.50 1.76 2.13 pPS16_009 clone 7 282.1 2.31 1.93 High Fidelity Phusion PCR of transformed cell with ligation 2 and 3 By Laetitia Phusion PCR was performed on the ligation product 2 (gBlock 2.1 ligated with gBblock 2.2) and the ligation product 3 (gBlock 3.1 ligated with gBlock 3.2) made on 09/08/16. It was done following this protocol. For the PCR on ligation 2 the primers used were IPS 123 and IPS 84. For the PCR on ligation 3 the primers used were IPS 128 and IPS 83. Gel1 DreamTaq PCR of transformed cell with pPS16_002,008,010, 011, 012, 013, 014 and puC19 By Léa and Laetitia PCR DreamTaq was performed on 6 clones of each transformed cells from 10/08/16. Each clone was spread and put in liquid culture with LB and Ampicillin. No PCR products were observed on the gel. Phusion PCR on PSB1C3 By Alice pSB1C3 was amplified with Phusion polymerase following this protocol. Two primers (iPS41 and iPS42) were used. Annealing temperature was 70.9°C. After amplification, 5 µL of each PCR products with loading dye and 10 µL of DNA ladder were placed on gel and migrated at 100v during 30 min. PCR products expected: Plasmids expected band size (bp) PSB1C3 2070 Migration of PSB1C3 amplification Result of the ligation 2-3 PCR. By Caroline We made the PCR again as the previous result was not satisfying. Ligation product were cultivate overnight. Result of the ligation 2-3 PCR Bringing DNA closer Extraction of DS-SPcasN- and DS-TDcasN- By Terrence The extraction was carried out with the Nucleobond Ax Kit. Extraction of DS-SPcasN- and DS-TDcasN- Nano Drop: Plasmid name Concentration (ng/µL) 260/230 260/280 DS-SPcasN- 185.46 2.23 1.79 DS-TDcasN 198.97 1.88 1.79
By Léa
Dh5a cells were transformed with pPS16_020 (GFP 1-9 ligated with pSB1C3 digested), or the linearized pSB1C3 (control), or not transformed (control) using the usual protocol.
By Alice
After colony screening PCR, 3 clones (clones 3, 7 and 8) seemed to have the insert of interest. Plasmids from these clones were extracted following this protocol. After extraction, plasmids were migrated on a gel.
Band size expected:
Nano Drop:
By Terrence
The extraction was carried out following the usual protocol.
By Laetitia
Phusion PCR was performed on the ligation product 2 (gBlock 2.1 ligated with gBblock 2.2) and the ligation product 3 (gBlock 3.1 ligated with gBlock 3.2) made on 09/08/16. It was done following this protocol. For the PCR on ligation 2 the primers used were IPS 123 and IPS 84. For the PCR on ligation 3 the primers used were IPS 128 and IPS 83.
Gel1
By Léa and Laetitia
PCR DreamTaq was performed on 6 clones of each transformed cells from 10/08/16. Each clone was spread and put in liquid culture with LB and Ampicillin. No PCR products were observed on the gel.
pSB1C3 was amplified with Phusion polymerase following this protocol. Two primers (iPS41 and iPS42) were used. Annealing temperature was 70.9°C. After amplification, 5 µL of each PCR products with loading dye and 10 µL of DNA ladder were placed on gel and migrated at 100v during 30 min.
PCR products expected:
By Caroline
We made the PCR again as the previous result was not satisfying. Ligation product were cultivate overnight.
The extraction was carried out with the Nucleobond Ax Kit.
