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 12th October 1.1 Visualization 1.1.1 Digestion of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 and GFP 1.9 PCR product 1.1.2 Gel of digested products 1.1.3 Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation 1.1.4 Cloning of GFP 1.9 from pUC19 (pPS16_009) in pSB1C3 by digestion-ligation 1.1.5 Transformation of DH5a cells with FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) and GFP 1.9 in pSB1C3 (pPS16_020) obtained by Digestion-Ligation 1.1.6 Liquid culture of FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) and GFP 1.9 in pSB1C3 transformed the 11th October Wednesday 12th October Visualization Digestion of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 and GFP 1.9 PCR product By Sylvie Extracted pPS16_019 and pPS16_018 from the 10 th October and the GFP 1.9 PCR product from the 4th October were digested by several restriction enzymes: 6 µL of FRB - GFP 11 in pSB1C3 (pPS16_019) 4 µL of buffer Tango 2 µL of restriction enzyme PstI 1 µL of restriction enzyme XbaI 1 µL of restriction enzyme NcoI 26 µL of water And: 2 µL of GFP 1.9 PCR product 2 µL of buffer Orange 1 µL of restriction enzyme NdeI 15 µL of water A control was done with SpeI and the mix were incubated for 2 hours at 37°C. Gel of digested products By Sylvie After digestion, 20 µL of digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. Result of the migration The results for GFP 1.9 could not be interpreted, the other results were good. Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation By Sylvie Once template and vector were cut, DNA ligase was used to join the sticky ends of the template and vector together: 11 µL of template (pPS16_019 treated by XbaI, NcoI & PstI) 5 µL of vector (pPS16_018 treated by SpeI) 2 µL of Buffer T4 10X 1 µL of ligase T4 enzyme 1 µL of water The mix was incubated for 30 minutes at rooming temperature. Cloning of GFP 1.9 from pUC19 (pPS16_009) in pSB1C3 by digestion-ligation By Sylvie Once template and vector were cut, DNA ligase was used to join the sticky ends of the template and vector together: 1 µL of template (GFP 1.9 treated by NdeI) 1 µL of vector (BbaB0015 treated by PstI & XbaI) 1 µL of Buffer T4 10X 1 µL of ligase T4 enzyme 6 µL of water The mix was incubated for 30 minutes at rooming temperature. Transformation of DH5a cells with FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) and GFP 1.9 in pSB1C3 (pPS16_020) obtained by Digestion-Ligation By Sylvie Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11 - FKBP - GFP 10 (pPS16_021), and with pSB1C3 containing GFP 1.9 (pPS16_020) or controls (digested plasmid) using the usual protocol. Liquid culture of FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) and GFP 1.9 in pSB1C3 transformed the 11th October By Sylvie Few clones were obtained for the transformation done yesterday. Clones were selected and put on liquid cultures (LB + 30 µg/mL Cm) grown at 37°C overnight.
By Sylvie
Extracted pPS16_019 and pPS16_018 from the 10 th October and the GFP 1.9 PCR product from the 4th October were digested by several restriction enzymes:
And:
A control was done with SpeI and the mix were incubated for 2 hours at 37°C.
After digestion, 20 µL of digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
The results for GFP 1.9 could not be interpreted, the other results were good.
Once template and vector were cut, DNA ligase was used to join the sticky ends of the template and vector together:
The mix was incubated for 30 minutes at rooming temperature.
Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11 - FKBP - GFP 10 (pPS16_021), and with pSB1C3 containing GFP 1.9 (pPS16_020) or controls (digested plasmid) using the usual protocol.
Few clones were obtained for the transformation done yesterday. Clones were selected and put on liquid cultures (LB + 30 µg/mL Cm) grown at 37°C overnight.