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 Thursday 7th July 1.1 Lab work 1.1.1 Visualization 1.1.1.1 PCR products migation 1.1.1.2 Cultures results 1.1.1.3 Culture of transformed pPS16_004, pPS16_007 and the pPS16_007 clone 6 1.1.2 caracterization 1.1.2.1 BL21 pre-culture 1.1.2.2 Cas9 PCR Thursday 7th July Lab work Visualization PCR products migation By Caroline and Léa gBlocks screened the 06/07/2016 were put on migration. The usual protocol was followed. 20µL of each PCR product was added to 4µL of purple loading dye 6X. PHOTO GEL pas dans le cahier ! Cultures results There was no blue colony observed on xGal/IPTG + ampicillin mediums for transformed transformed pPS16_004, pPS16_007 and the pPS16_007 clone 6 gBlocks. Culture of transformed pPS16_004, pPS16_007 and the pPS16_007 clone 6 By Laetitia and Charlène Because of the absence of blue colony after culture, xGal and IPTG efficency was tested. A petri dishe was splitted in four parts : new xGal, new IPTG new xGal, former IPTG former xGal, new IPTG former xGal, former IPTG A blue colony from the control tube 2 of the 04/07/2016 was plated on each of the four part on medium LB + Ampicillin (100µg/mL) + xGal/IPTG (1/1000). 100µ of pPS16_004, pPS16_007 or the pPS16_007 clone 6 bacteries were plated again on LB + Ampicillin (100µg/mL) + xGal/IPTG (1/1000) medium. caracterization BL21 pre-culture By Laetitia and Charlène A BL21 colony was added to 4µL of LB and put in incubation overnight at 37°c, 200 rpm. Cas9 PCR By Léa Forward primer Sequencing addgene Cas plasmids (IPS 134) and Reverse primer Sequencing addgene Cas plasmids (IPS 135) were used for NM Cas9(DS-NMcas), ST1 Cas9 (DS-ST1casN-) and Td Cas9 (DS-TDcasN-). For SP Cas9 (DS-SPcasN-) we used primers : IPS 134 + IPS 136 = SP1 IPS 137 + IPS 135 = SP2 The PCR was conducted following the usual protocol
By Caroline and Léa
gBlocks screened the 06/07/2016 were put on migration. The usual protocol was followed. 20µL of each PCR product was added to 4µL of purple loading dye 6X.
PHOTO GEL pas dans le cahier !
There was no blue colony observed on xGal/IPTG + ampicillin mediums for transformed transformed pPS16_004, pPS16_007 and the pPS16_007 clone 6 gBlocks.
By Laetitia and Charlène
Because of the absence of blue colony after culture, xGal and IPTG efficency was tested. A petri dishe was splitted in four parts :
A blue colony from the control tube 2 of the 04/07/2016 was plated on each of the four part on medium LB + Ampicillin (100µg/mL) + xGal/IPTG (1/1000).
100µ of pPS16_004, pPS16_007 or the pPS16_007 clone 6 bacteries were plated again on LB + Ampicillin (100µg/mL) + xGal/IPTG (1/1000) medium.
A BL21 colony was added to 4µL of LB and put in incubation overnight at 37°c, 200 rpm.
By Léa
Forward primer Sequencing addgene Cas plasmids (IPS 134) and Reverse primer Sequencing addgene Cas plasmids (IPS 135) were used for NM Cas9(DS-NMcas), ST1 Cas9 (DS-ST1casN-) and Td Cas9 (DS-TDcasN-). For SP Cas9 (DS-SPcasN-) we used primers :
The PCR was conducted following the usual protocol
