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 5th September 1.1 Lab work 1.1.1 Visualization 1.1.1.1 fragments 1.1 with 1.2 Ligation AND 2.1 with 2.2 Ligation 1.1.1.2 Clean-up of ligation products 1.1.1.3 Q5 PCR and phusion PCR on ligation product 1.1.1.4 Gibson of cleaned up PCR products fragment 1 and fragment 2 in pSB1C3 treated by DpnI Monday 5th September Lab work Visualization fragments 1.1 with 1.2 Ligation AND 2.1 with 2.2 Ligation By Mahnaz Fragment 1.1 and 1.2 were ligated together as following to create fragment 1 4µL of Fragment 1.1 PCR product from the 02/09/2016 4µM of Fragment 1.2 PCR product from 02/09/2016 1µL of Buffer T4 10X 1µL of ligase T4 enzyme Fragment 2.1 and 2.2 were ligated together as following to create fragment 2 4µL of Fragment 2.1 PCR product from the 02/09/2016 4µM of Fragment 2.2 PCR product from 02/09/2016 1µL of Buffer T4 10X 1µL of ligase T4 enzyme The ligation product was put at room temperature for 1 hour. Clean-up of ligation products By Mahnaz Ligation product (fragement 1 and fragment 2) obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol. Q5 PCR and phusion PCR on ligation product By Mahnaz Q5 and phusion PCR was performed on the ligation product (fragment 1 and fragment 2) 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 Fragment 1 Fragment 2 Primers iPS122 and iPS140 iPS123 and iPS84 Tm 72°C 72°C t 1 min 1 min Result of migration The PCR product of ligation is not in high concentration (mostly with phusion polymerase) Following Gibson assembly was done by both fragments. Gibson of cleaned up PCR products fragment 1 and fragment 2 in pSB1C3 treated by DpnI By Manhaz Gibson was performed with cleaned up PCR products fragment1 x fragment 2 x pSB1C3 treated by DpnI (plasmid) with the following protocol: For each 20μl of reaction, mix the following reagents :
By Mahnaz
Fragment 1.1 and 1.2 were ligated together as following to create fragment 1
Fragment 2.1 and 2.2 were ligated together as following to create fragment 2
The ligation product was put at room temperature for 1 hour.
Ligation product (fragement 1 and fragment 2) obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
Q5 and phusion PCR was performed on the ligation product (fragment 1 and fragment 2) 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:
The PCR product of ligation is not in high concentration (mostly with phusion polymerase) Following Gibson assembly was done by both fragments.
By Manhaz
Gibson was performed with cleaned up PCR products fragment1 x fragment 2 x pSB1C3 treated by DpnI (plasmid) with the following protocol:
For each 20μl of reaction, mix the following reagents :
