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 24th August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Gel Electrophoresis of Gibson Assembly(fragment 3-4) and GFP 1-9 cloned in PSB1C3 1.1.1.2 pPS16_002 (1.2) clones 8 and 14 and pPS16_014(SgRna Nm) clone 12 Extraction 1.1.1.3 Transformation of gblockFRB in pJET and gblock Sg_RNA_NM in pJET 1.1.1.4 Plasmid DNA extraction (Midi Prep) 1.1.1.5 PCR Q5 on plasmid Wednesday 24th August Lab work Visualization Gel Electrophoresis of Gibson Assembly(fragment 3-4) and GFP 1-9 cloned in PSB1C3 By Terrence Colony PCR of 12 colonies containing the Gibson Assembly's products (fragment 3-4). Colony PCR of 12 colonies containing the GFP 1-9. - Clones 2, 4, 7 and 11 are as expected. pPS16_002 (1.2) clones 8 and 14 and pPS16_014(SgRna Nm) clone 12 Extraction By Terrence The extraction was carried out following the usual protocol. Result of the extraction Nano drop : size (ng/µL) 260/230 260/280 1.2.14 47.81 1.64 1.84 1.2.8 834.5 2.40 1.99 SgRna Nm 12 124.21 2.00 1.89 Transformation of gblockFRB in pJET and gblock Sg_RNA_NM in pJET By Terrence First we made a ligation with Component Volume (µL) Reaction Buffer 10 PCR Product (FRB or Nm) 1 pJET1.2/blunt 1 water, nuclease free 7 T4 DNA Ligase 1 Then we transformed 2µL of ligation product into 50 µL of DH5a competent cells, following the usual protocol. Transformation products were plated with three different volume : 50µL , 150µl and 350µL. 350µL only for the control. Plasmid DNA extraction (Midi Prep) By Léa & Manhaz 200 mL of transformed DH5a cells with Adgene dCas9 Nm plasmids (clone 1 and 2) cultures were extracted usind a Midi Prep kit. It was resuspended into 50µL of sterile water. PCR Q5 on plasmid By Léa & Manhaz
By Terrence
- Clones 2, 4, 7 and 11 are as expected.
The extraction was carried out following the usual protocol.
Nano drop :
First we made a ligation with
Then we transformed 2µL of ligation product into 50 µL of DH5a competent cells, following the usual protocol.
Transformation products were plated with three different volume : 50µL , 150µl and 350µL. 350µL only for the control.
By Léa & Manhaz
200 mL of transformed DH5a cells with Adgene dCas9 Nm plasmids (clone 1 and 2) cultures were extracted usind a Midi Prep kit. It was resuspended into 50µL of sterile water.
