Friday 2^nd 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:

Primers used were:

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