Difference between revisions of "Team:Paris Saclay/Notebook/August/16"

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(Lab work)
 
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{{Team:Paris_Saclay/notebook_header}}
 
{{Team:Paris_Saclay/notebook_header}}
 
= Tuesday 16<sup>th</sup> August=
 
= Tuesday 16<sup>th</sup> August=
==Lab work==
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===Visualization===
 
===Visualization===
 
==== 2.1-2.2 and 3.1-3.2 ligation ====
 
==== 2.1-2.2 and 3.1-3.2 ligation ====
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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.
 
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====
 
==== Q5 PCR on the ligation products and pPS16_008 clones 1 and 2====
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[[File:T--Paris Saclay--20160816 fragments 2, 3 and gblock 4.1.JPG|500px|thumb|right|Gel Electrophoresis of PCR products fragments 2, 3 and gblock 4.1.JPG]]
 
[[File:T--Paris Saclay--20160816 fragments 2, 3 and gblock 4.1.JPG|500px|thumb|right|Gel Electrophoresis of PCR products fragments 2, 3 and gblock 4.1.JPG]]
  
 
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All the bands have the good size so the PCR products were purify
All the bands have the good size so purifications will be done on PCR products.
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==== PCR Clean-up with the NucleoSpin kit ====
 
==== PCR Clean-up with the NucleoSpin kit ====
 
''By Mahnaz''
 
''By Mahnaz''
 
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_
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]].
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The purification was carried out on PCR products pSB1C3 (linearized) and 4.2 also the ligation products pPS16_003-pPS16_004 and pPS16_005-pPS16_006 following the usual [[Team:Paris_Saclay/Experiments#Purification|protocol]].
  
 
[[File:T--Paris_Saclay--180816_visualization_PCR_CleanUp.jpg|400px|thumb|right|Result of the PCR]]
 
[[File:T--Paris_Saclay--180816_visualization_PCR_CleanUp.jpg|400px|thumb|right|Result of the PCR]]

Latest revision as of 16:42, 9 October 2016

Tuesday 16th August

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.

Gel Electrophoresis of PCR products fragments 2, 3 and gblock 4.1.JPG

All the bands have the good size so the PCR products were purify

PCR Clean-up with the NucleoSpin kit

By Mahnaz _ The purification was carried out on PCR products pSB1C3 (linearized) and 4.2 also the ligation products pPS16_003-pPS16_004 and pPS16_005-pPS16_006 following the usual protocol.

Result of the PCR

4.1-4.2 ligation

By Mahnaz

8 µL of 4.1 purified PCR product, 8µL of 4.2 purified PCR product, 2 µL of ligase T4 Buffer and 2 µL of ligase T4 were mixed. The ligation reaction mixture were incubated overnight at 4°C.