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 Sunday 2nd October 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Digestion of GFP 1.9 PCR product from the 9th September 1.1.1.2 Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation 1.1.1.3 Cloning of GFP 1.9 from pUC19 (pPS16_009) in pSB1C3 by digestion-ligation 1.1.1.4 Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 and GFP 1.9 in pUC19 (pPS16_009) clone 1 Sunday 2nd October Lab work Visualization Digestion of GFP 1.9 PCR product from the 9th September By Maxence & Victor As we had several issues to clones GFP 1.9 in pSB1C3 by Gibson and digestion-ligation, GFP 1.9 PCR product from the 9th September was digested by restriction NdeI enzymes in order to verify if the template we used was the good one. For that purpose, GFP 1.9 PCR product from the 9th September was cut by restriction enzymes NdeI as following: 2 µL of GFP 1.9 PCR product from the 9th September 1 µL of buffer orange 1 µL of restriction enzyme NdeI 6 µL of water The mix was incubated for 30 minutes at 37°C. Then, 20 µL of each digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation By Maxence & Victor Once template and vector were cut, they were mix together with water and were precipitated by ethanol as they were not concentrated enough. The ethanol precipitation was run as following: 28 µL of template (pPS16_019 treated by XbaI & PstI) 13 µL of vector (pPS16_018 treated by SpeI & PstI) 9 µL of water 50 µL of isopropanol 5 µL of CH<inf>3</inf>COONa DNA ligase was used to join the sticky ends of the template and vector together: 1.5 µL of Buffer T4 10X 1 µL of ligase T4 enzyme 12.5 µL of water The mix were incubated for 30 minutes at rooming temperature. Cloning of GFP 1.9 from pUC19 (pPS16_009) in pSB1C3 by digestion-ligation By Maxence & Victor Once template and vector were cut, DNA ligase was used to join the sticky ends of the template and vector together: 6 µL of template (pPS16_009 treated by PstI & XbaI) 3 µL of vector (Bba treated by PstI & XbaI) 1.5 µL of Buffer T4 10X 1 µL of ligase T4 enzyme 3.5 µL of water The mix were incubated for 30 minutes at rooming temperature. Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 and GFP 1.9 in pUC19 (pPS16_009) clone 1 "By Maxence & Victor" The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit: pPS16_018 (FKBP - GFP 10) clone 6 pPS16_019 (FRB - GFP 11) clone 4 pPS16_009 (GFP 1.9) clone 1
By Maxence & Victor
As we had several issues to clones GFP 1.9 in pSB1C3 by Gibson and digestion-ligation, GFP 1.9 PCR product from the 9th September was digested by restriction NdeI enzymes in order to verify if the template we used was the good one.
For that purpose, GFP 1.9 PCR product from the 9th September was cut by restriction enzymes NdeI as following:
The mix was incubated for 30 minutes at 37°C. Then, 20 µL of each digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
Once template and vector were cut, they were mix together with water and were precipitated by ethanol as they were not concentrated enough. The ethanol precipitation was run as following:
DNA ligase was used to join the sticky ends of the template and vector together:
The mix were incubated for 30 minutes at rooming temperature.
Once template and vector were cut, DNA ligase was used to join the sticky ends of the template and vector together:
"By Maxence & Victor"
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
