Monday 5^th 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:

Primers used were:

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.