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+ [[File:T--Paris_saclay--0209.extension.png|400px|thumb|center|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 ====
====PCR Clean-up of PCR products ====
Revision as of 10:17, 23 September 2016
Friday 2th th 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
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
