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 Monday 22th August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Low fidelity Dreamn Taq PCR of pPS16_016 1.1.1.2 Sequencing of 1.2, FKBP, FRB 1.1.1.3 Purification on gel of gBlocks 3 and 4 1.1.1.4 Purification of PSB1C3 with kit 1.1.1.5 Gibson assembly product transformation in DH5a cells 1.1.1.6 pPS16_016 ligation product transformation in DH5a 1.1.2 Interlab Study 1.1.2.1 Transformation into DH5a cells 1.1.2.2 Device 1 culture Monday 22th August Lab work Visualization Low fidelity Dreamn Taq PCR of pPS16_016 By Mathilde Clones 7,8,9 and 10 of pPS16_016 were amplified following the protocol : 1,5µL of Green Buffer 1µL of dNTPs 1µL of each primers IPS83 and IPS84 0,13µL of Dream Taq Polymerase 19,37 µL of H20 Colonies were put into sterile water, put through 5 minutes of denaturation before the addition of the mix. Annealing temperature was 57°c. After amplification, 1µL of each PCR product and 10µL of the DNA purple ladder were palced in wells and migrated at 100V for 30min. Result of the PCR Sequencing of 1.2, FKBP, FRB Terrence and Léa Was sent : Clone Concentration size 1.2 11 392.66 3934 FKBP 3 272.37 3393 FKBP 4 404.65 3393 FKBP 5 204.87 3393 FRB 2 256.44 3347 FRB 5 129.86 3347 Purification on gel of gBlocks 3 and 4 Terrence and Mathilde After amplification, 50µL of each PCR product diluted 6 tumes with 10 µL of the gel loadding dye, and 10µL of the DNA purple ladder were placed in wells and migrated at 100V for 30min. The extraction was carried out with the usual protocol. Nano Drop : size (ng/µL) 260/230 260/280 Fragment 3 11.07 0.48 3.02 Fragment 4 50.12 1.07 1.93 Gel for the extraction Purification of PSB1C3 with kit Terrence PSB1C3 was purified following the usual protocol. To be sure that PSB1C3 were linear, it was digested with DPN1. Nano Drop : size (ng/µL) 260/230 260/280 PSB1C3 58.66 0.74 1.71 Gibson assembly product transformation in DH5a cells by Léa Gibson assembly product (fragments 3 and 4 ligation) and control were transformed into DH5a cells, using 2 µL of DNA, and following the usual protocol. pPS16_016 ligation product transformation in DH5a By Léa pPS16_016 ligation product of the 10/08/2016 was transformed into DH5a cells, using 2 µL of DNA, and following the usual protocol. Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C ON. Interlab Study Transformation into DH5a cells By Léa Devices 2 and 3, cnegative control and positive control was transformed into DH5a cells, following the usual protocol. Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C ON. Device 1 culture By Léa Device 1 transformed DH5a glycerol stock was plated on LB Agar chloramphenicol Petri Dishes and incubated at 37°C ON.
By Mathilde
Clones 7,8,9 and 10 of pPS16_016 were amplified following the protocol :
Colonies were put into sterile water, put through 5 minutes of denaturation before the addition of the mix. Annealing temperature was 57°c. After amplification, 1µL of each PCR product and 10µL of the DNA purple ladder were palced in wells and migrated at 100V for 30min.
Terrence and Léa
Was sent :
Terrence and Mathilde
After amplification, 50µL of each PCR product diluted 6 tumes with 10 µL of the gel loadding dye, and 10µL of the DNA purple ladder were placed in wells and migrated at 100V for 30min. The extraction was carried out with the usual protocol.
Nano Drop :
Terrence
PSB1C3 was purified following the usual protocol. To be sure that PSB1C3 were linear, it was digested with DPN1.
by Léa
Gibson assembly product (fragments 3 and 4 ligation) and control were transformed into DH5a cells, using 2 µL of DNA, and following the usual protocol.
By Léa
pPS16_016 ligation product of the 10/08/2016 was transformed into DH5a cells, using 2 µL of DNA, and following the usual protocol. Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C ON.
Devices 2 and 3, cnegative control and positive control was transformed into DH5a cells, following the usual protocol. Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C ON.
Device 1 transformed DH5a glycerol stock was plated on LB Agar chloramphenicol Petri Dishes and incubated at 37°C ON.
