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 2nd August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Q5 PCR and Phusion PCR of gblock 1.1 in pUC19, gblock 1.2 in pJET , gblock 2.1 in pUC19 and gblock 2.2 in pUC19 1.1.1.2 PCR Clean-up of PCR products 1.1.1.3 NanoDrop Measurements Friday 2nd August Lab work Visualization Q5 PCR and Phusion PCR of gblock 1.1 in pUC19, gblock 1.2 in pJET , gblock 2.1 in pUC19 and gblock 2.2 in pUC19 By Mahnaz Q5 PCR and phusion PCR were performed on plasmids with the following protocol: For each 50μl of reaction, mix the following reagents: 1 µL of plasmid 1 µL of dNTPs (10mM) 1 µL of each primer mix (10µM) 10 µL of Q5 buffer (5X) 0,5 µL of Q5 high fidelity polymerase 35,5 µL of nuclease free water Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow: Step Temperature Time Initial denaturation 98°C 30sec 30 cycles 98°C 10sec Tm 20sec 72°C t Final Extension 72°C 2min Hold 4°C infinity Primers used were: Matrix gblock 1.1 in pUC19 gblock 1.2 in pJET gblock 2.1 in pUC19 gblock 2.2 in pUC19 Primers iPS140 and iPS120 iPS121 and iPS122 iPS123 and iPS124 iPS125 and iPS84 Tm 72°C 72°C 72°C 72°C t 30 sec 30 sec 30 sec 30 sec Result of migration This experiment confirms the efficiency of Q5 enzyme in amplifying the gblocks considering the annealing temperature of the primers which are rather high 72°C. PCR Clean-up of PCR products By Mahnaz PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol. NanoDrop Measurements By Mahnaz Sample PCR reaction Q5 reaction Concentration (ng/µL) phusion reaction Concentration (ng/µL) gblock 1.1 161.88 263.81 gblock 1.2 218.01 209.24 gblock 2.1 101.16 185.47 gblock 2.2 113.88 138.87
By Mahnaz
Q5 PCR and phusion PCR were performed on plasmids with the following protocol:
For each 50μl of reaction, mix the following reagents:
Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:
Primers used were:
This experiment confirms the efficiency of Q5 enzyme in amplifying the gblocks considering the annealing temperature of the primers which are rather high 72°C.
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
