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 Monday 4th July 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Ligation of Gblock 2-2 1.1.1.2 Transformation of Gblocks in DH5α 1.1.1.3 Digestion of plasmids 1.1.2 Bringing DNA closer 1.1.2.1 Plasmids extraction Monday 4th July Lab work Visualization Ligation of Gblock 2-2 By Caroline and Lea TODOà remplir Transformation of Gblocks in DH5α By Caroline and Lea 6 transformations were performed with the ligation products containing plasmids: pPS16_001, pPS16_002, pPS16_003, pPS16_004, pPS16_005, pPS16_006. 3 other transformations were performed with plasmids pPS16_007, pPS16_008 and pPS16_009 extracted the 30/06/16. TODOajouter le lien The transformation protocol were followed increasing plasmids quantities (5µL instead of 1µL). After transformation, cells are spread on petri dishes containing LB + Ampicillin (50µg/mL) + Xgal + IPTG. Digestion of plasmids By Mathilde and Alice The following plasmids were digested again with EcoR1 and HindIII using this protocol: pPS16_001 (from the 6 clones that were selected on 29/06/16) pPS16_003 (from the 6 clones that were selected on 29/06/16) pPS16_005 (from the 6 clones that were selected on 29/06/16) pPS16_006 (from the 6 clones that were selected on 29/06/16) Component Volume (µL) Plasmid 10 Red buffer 10x 2 Water 7 EcoRI enzyme 0.5 HindIII enzyme 0.5 The digestion products were mixed as follow to be migrated on an agarose gel. Component Volume (µL) Digested DNA 20 Loading buffer 3.3 The DNA concentration was still insufficient meaning the extraction step was not efficient. Bringing DNA closer Plasmids extraction By Naiane and Laetitia Plasmids from the following strains were extracted with the kit "Charge Switch-Pro Plasmid Miniprep": DS-NMcas DS-SPcasN- DS-ST1casN- DS-TDcasN- Then plasmids were digested with AvrII Component Volume (µL) Plasmid 10 Tango buffer 10x 2 Water 7 AvrII enzyme 1 The mix was incubated at 37°C for 1 hour. An electrophoresis was done (0.8% of agarose). Component Volume (µL) Digested DNA 5 Water 5 Loading buffer 2
By Caroline and Lea
TODOà remplir
6 transformations were performed with the ligation products containing plasmids: pPS16_001, pPS16_002, pPS16_003, pPS16_004, pPS16_005, pPS16_006. 3 other transformations were performed with plasmids pPS16_007, pPS16_008 and pPS16_009 extracted the 30/06/16. TODOajouter le lien The transformation protocol were followed increasing plasmids quantities (5µL instead of 1µL). After transformation, cells are spread on petri dishes containing LB + Ampicillin (50µg/mL) + Xgal + IPTG.
By Mathilde and Alice
The following plasmids were digested again with EcoR1 and HindIII using this protocol:
The digestion products were mixed as follow to be migrated on an agarose gel.
The DNA concentration was still insufficient meaning the extraction step was not efficient.
By Naiane and Laetitia
Plasmids from the following strains were extracted with the kit "Charge Switch-Pro Plasmid Miniprep":
Then plasmids were digested with AvrII
The mix was incubated at 37°C for 1 hour. An electrophoresis was done (0.8% of agarose).
