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 Friday 26th August 1.1 Visualization 1.1.1 Purification of pPS16-003 and pPS16_004 ligation products 1.1.2 Q5 high fidelity PCR of pPS16_003 - pPS16_004 ligation product 1.1.3 Purification of fragments 1(16-001 + pPS16_002) and 2 (16-003 + pPS16_004) 1.1.4 Gibson Assembly of segments 1 and 2 1.1.5 Transformation in DH5α with the 1-2 Gibson assembly product Friday 26th August Visualization Purification of pPS16-003 and pPS16_004 ligation products By Mathilde Ligation products pPS16-003 and pPS16_004 frome the day before were purificated according to the usual protocol Q5 high fidelity PCR of pPS16_003 - pPS16_004 ligation product By Mathilde The Q5 PCR was conducted following the exact same protocol than the 25/08/2016 on the ligation product from the day before. Migration Results The two bands are at the expected size (1860 pb), so the fragment 2 (16-003 and pPS16_004 ligation product) is purified. Purification of fragments 1(16-001 + pPS16_002) and 2 (16-003 + pPS16_004) By Mathilde The purification was made following the usual protocol. Each of those purification products were quantified on nanodrop. Segment DNA quantity (ng/µL) 260/230 260/280 1 84,20 1,25 1,84 2 169,88 1,29 1,83 Enough DNA quantity was obtained to perform a Gibson assembly on segments 1 and 2. Gibson Assembly of segments 1 and 2 By mathilde Two preparations were made : the 1-2 Gibson assembly product tube : 1,12µL of purified segment 1 and 0,56µL of purified segment 2 + 0,85µL of digested pSB1C3 + 7,47 µL of sterile water + 10µL of NEB Builder Hifi DNA Assembler Master Mix the control tube : 0,85µL of digested pSB1C3 + 17,47 µL of sterile water Gibson products were incubated 1h at 52°c. Transformation in DH5α with the 1-2 Gibson assembly product By Mathilde The transformation was performed following the usual protocol with 2µL of plasmid. 50, 150 and 350µL of the transformed DH5α with the 1-2 Gibson assembly product were plated on 3 plates of LB + chloramphenicol (30µg/mL) 350 µL of each Gibson assembly control and transformation control were plated on 2 plates of B + chloramphenicol (30µg/mL) Petri cultures were incubated at 37°c overnight.
By Mathilde
Ligation products pPS16-003 and pPS16_004 frome the day before were purificated according to the usual protocol
The Q5 PCR was conducted following the exact same protocol than the 25/08/2016 on the ligation product from the day before.
The two bands are at the expected size (1860 pb), so the fragment 2 (16-003 and pPS16_004 ligation product) is purified.
The purification was made following the usual protocol. Each of those purification products were quantified on nanodrop.
Enough DNA quantity was obtained to perform a Gibson assembly on segments 1 and 2.
By mathilde
Two preparations were made :
Gibson products were incubated 1h at 52°c.
The transformation was performed following the usual protocol with 2µL of plasmid.
Petri cultures were incubated at 37°c overnight.
