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 Thuersday 22nd September 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Glycerol stocks of clones X, X, X and X containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) 1.1.1.2 Plasmids extraction of clones X, X, X and X containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) 1.1.1.3 Digestion of plasmids extracted from clones X, X, X and X containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) 1.1.1.4 Colony PCR of 16 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022) Thuersday 22nd September Lab work Visualization Glycerol stocks of clones X, X, X and X containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) "By Maxence, Mahnaz & Coline" The glycerol stock of the bacteria with the following plasmids were made. pPS16_017 (dCas9 ST - GFP 11) clone X pPS16_017 (dCas9 ST - GFP 11) clone X pPS16_017 (dCas9 ST - GFP 11) clone X pPS16_017 (dCas9 ST - GFP 11) clone X 500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C. Plasmids extraction of clones X, X, X and X containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) "By Maxence, Mahnaz & Coline" The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit: pPS16_017 (dCas9 ST - GFP 11) clone X pPS16_017 (dCas9 ST - GFP 11) clone X pPS16_017 (dCas9 ST - GFP 11) clone X pPS16_017 (dCas9 ST - GFP 11) clone X Digestion of plasmids extracted from clones X, X, X and X containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) "By Maxence, Mahnaz & Coline" To assess if DH5a cells were transformed with the good plasmid, plasmid digestion was run with XbaI as following: 1 µL of extracted plasmid 1 µL of buffer 1 µL of restriction enzyme XbaI 7 µL of water A control was done with 1 µL of non-digested plasmid. GEL Colony PCR of 16 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022) By Maxence, Mahnaz & Coline A colony PCR was done for 16 clones from the 20th September. For that purpose, 16 clones were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C. For each clones contained in 20 μl water, 5.13 μL of the following mix were added : 2.5 µL DreamTaq Buffer 0.5 µL of dNTPs (10mM) 1 µL of each primer mix (10µM) 0.13 μl of DreamTaq Pol PCR was performed as follow: Step Temperature Time Initial denaturation 95°C 3 min 30 cycles 95°C 30 sec 48.4°C 30 sec 72°C 1min 30sec Final Extension 72°C 7 min Hold 4°C $\infty$ Primers used were: Matrix Clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022) Primers iPS168 and iPS169 After amplification, 3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. PCR products expected were : PCR products Expected band size (bp) FRB - GFP 11 - GFP 1.9 1500 GEL
"By Maxence, Mahnaz & Coline"
The glycerol stock of the bacteria with the following plasmids were made.
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
To assess if DH5a cells were transformed with the good plasmid, plasmid digestion was run with XbaI as following:
A control was done with 1 µL of non-digested plasmid.
GEL
By Maxence, Mahnaz & Coline
A colony PCR was done for 16 clones from the 20th September. For that purpose, 16 clones were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.
For each clones contained in 20 μl water, 5.13 μL of the following mix were added :
PCR was performed as follow:
Primers used were:
After amplification, 3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
PCR products expected were :
