Difference between revisions of "Team:Paris Saclay/Notebook/September/2"

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{{Team:Paris_Saclay/notebook_header}}
 
{{Team:Paris_Saclay/notebook_header}}
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= Friday 2<sup>th</sup> September=
+
 
−
==Lab work==
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=Friday 2<sup>nd</sup> August=
 +
 
 
===Visualization===
 
===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:
 +
{| class="wikitable"
 +
|-
 +
!Step
 +
!Temperature
 +
!Time
 +
|-
 +
|Initial denaturation
 +
|98°C
 +
|30sec
 +
|-
 +
|rowspan="3"|30 cycles
 +
|98°C
 +
|10sec
 +
|-
 +
|Tm
 +
|20sec
 +
|-
 +
|72°C
 +
|t
 +
|-
 +
|Final Extension
 +
|72°C
 +
|2min
 +
|-
 +
|Hold
 +
|4°C
 +
|infinity
 +
|}
 +
 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 +
 +
{| class="wikitable"
 +
|-
 +
!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
 +
|}
 +
 +
[[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 ====
 +
''By Mahnaz''
 +
 +
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]].
  
 +
====NanoDrop Measurements====
 +
''By Mahnaz''
  
 +
{| class="wikitable"
 +
!Sample PCR reaction
 +
! Q5 reaction Concentration (ng/µL)
 +
! phusion reaction Concentration (ng/µL)
 +
|-
 +
|gblock 1.1 <div id=" gblock 1.1 "></div>
 +
|161.88
 +
|263.81
 +
|-
 +
| gblock 1.2 <div id=" gblock 1.2 "></div>
 +
|218.01
 +
|209.24
 +
|-
 +
| gblock 2.1 <div id=" gblock 2.1 "></div>
 +
|101.16
 +
|185.47
 +
|-
 +
| gblock 2.2 <div id=" gblock 2.2 "></div>
 +
|113.88
 +
|138.87
 +
|-
 +
|}
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 17:02, 9 October 2016

Friday 2nd August

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
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

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





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