Line 72:
Line 72: ''By Mahnaz''
''By Mahnaz''
− The purification was carried out on PCR product PSB1C3 and 4.2 also the ligation products PS16003-PS16004 and PS16005-PS16006 following the usual [[Team:Paris_Saclay/Experiments#Purification|protocol]].
+ The purification was carried out on PCR product PSB1C3 (linearized) and 4.2 also the ligation products PS16003-PS16004 and PS16005-PS16006 following the usual [[Team:Paris_Saclay/Experiments#Purification|protocol]].
==== 4.1-4.2 ligation ====
==== 4.1-4.2 ligation ====
Revision as of 08:46, 18 August 2016
Tuesday 16th August Lab work Visualization 2.1-2.2 and 3.1-3.2 ligation By Charlène
8µL of 2.1 purify PCR products, 8µL of 2.2 purify PCR products, 2µL of ligase T4 Buffer and 2L of ligase T4 were mixed. 8µL of 3.1 purified PCR products, 8µL of 3.2 purified PCR products, 2µL of ligase T4 Buffer and 2µL of ligase T4 were mixed. They were incubated for 1h at RT.
Q5 PCR on the ligation products and pPS16_008 clones 1 and 2 By Charlène
The PCR was carried out with a new protocol:
Q5 PCR recipe
Buffer Q5 HF (5X)
10µL
dNTP (10mM)
1µL
Primers (10 µM, each)
2,5µL
DNA
1µL for plasmid, 2µL for ligation's products
Q5 DNA polymerase
0.25µL
Nuclease-free water
up to 50µL
Steps for PCR :
Step
Temperature
Time
Initial denaturation
98°C
30sec
30 cycles
98°C
5sec
Tannealing
30sec
72°C
30sec/kb
Final Extension
72°C
2min
Hold
4°C
$\infty$
An specific primers for each part was used.
The products were migrated on a 0.8% agarose gel with BET.
PCR Clean-up with the NucleoSpin kit By Mahnaz
The purification was carried out on PCR product PSB1C3 (linearized) and 4.2 also the ligation products PS16003-PS16004 and PS16005-PS16006 following the usual protocol .
4.1-4.2 ligation By Mahnaz
8µL of 4.1 purified PCR products, 8µL of 4.2 purified PCR products, 2µL of ligase T4 Buffer and 2L of ligase T4 were mixed. The ligation reaction mixture were incubated for overnight in the 4°c.
