Difference between revisions of "Team:Paris Saclay/Notebook/September/5"

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(→‎Gibson of cleaned up PCR products fragment 1 and fragment 2 in pSB1C3 treated by DpnI)
 
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==Lab work==
 
==Lab work==
 
===Visualization===
 
===Visualization===
 +
 +
====fragments 1.1 with 1.2 Ligation AND 2.1 with 2.2 Ligation====
 +
''By Mahnaz''
 +
 +
Fragment 1.1 and 1.2 were ligated together as following to create fragment 1
 +
* 4µL of Fragment 1.1 PCR product from the 02/09/2016
 +
* 4µM of Fragment 1.2 PCR product from 02/09/2016
 +
* 1µL of Buffer T4 10X
 +
* 1µL of ligase T4 enzyme
 +
 +
Fragment 2.1 and 2.2 were ligated together as following to create fragment 2
 +
* 4µL of Fragment 2.1 PCR product from the 02/09/2016
 +
* 4µM of Fragment 2.2 PCR product from 02/09/2016
 +
* 1µL of Buffer T4 10X
 +
* 1µL of ligase T4 enzyme
 +
 +
The ligation product was put at room temperature for 1 hour.
 +
 +
==== Clean-up of ligation products ====
 +
''By Mahnaz''
 +
 +
Ligation product (fragement 1 and fragment 2) obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]].
 +
 +
====Q5 PCR and phusion PCR on ligation product ====
 +
''By Mahnaz''
 +
 +
Q5 and phusion PCR was performed on the ligation product (fragment 1 and fragment 2) 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
 +
! Fragment 1
 +
! Fragment 2
 +
|-
 +
|Primers
 +
|iPS122 and iPS140
 +
|iPS123 and iPS84
 +
|-
 +
|Tm
 +
|72°C
 +
|72°C
 +
|-
 +
|t
 +
|1 min
 +
|1 min
 +
|}
 +
 +
[[File:T--Paris_saclay--0509.extension.png|400px|thumb|center|Result of migration ]]
 +
The PCR product of ligation is not in high concentration (mostly with phusion polymerase)
 +
Following Gibson assembly was done by both fragments.
 +
 +
====Gibson of cleaned up PCR products fragment 1 and fragment 2  in pSB1C3 treated by DpnI====
 +
''By Manhaz ''
 +
 +
Gibson was performed with cleaned up PCR products fragment1 x fragment 2 x pSB1C3 treated by DpnI (plasmid) with the following protocol:
 +
 +
For each 20μl of reaction, mix the following reagents :
 +
 +
* the 1-2 Gibson assembly product tube : 1,12µL of purified segment 1 and 0,56µL of purified segment 2 +  0,85µL of digested pSB1C3 + 7,47 µL of sterile water + 10µL of NEB Builder Hifi DNA Assembler Master Mix
 +
* the control tube : 0,85µL of digested pSB1C3 + 17,47 µL of sterile water
 +
 +
Gibson products were incubated 1h at 52°c.
  
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 12:39, 17 October 2016

Monday 5th September

Lab work

Visualization

fragments 1.1 with 1.2 Ligation AND 2.1 with 2.2 Ligation

By Mahnaz

Fragment 1.1 and 1.2 were ligated together as following to create fragment 1

  • 4µL of Fragment 1.1 PCR product from the 02/09/2016
  • 4µM of Fragment 1.2 PCR product from 02/09/2016
  • 1µL of Buffer T4 10X
  • 1µL of ligase T4 enzyme

Fragment 2.1 and 2.2 were ligated together as following to create fragment 2

  • 4µL of Fragment 2.1 PCR product from the 02/09/2016
  • 4µM of Fragment 2.2 PCR product from 02/09/2016
  • 1µL of Buffer T4 10X
  • 1µL of ligase T4 enzyme

The ligation product was put at room temperature for 1 hour.

Clean-up of ligation products

By Mahnaz

Ligation product (fragement 1 and fragment 2) obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.

Q5 PCR and phusion PCR on ligation product

By Mahnaz

Q5 and phusion PCR was performed on the ligation product (fragment 1 and fragment 2) 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 Fragment 1 Fragment 2
Primers iPS122 and iPS140 iPS123 and iPS84
Tm 72°C 72°C
t 1 min 1 min
Result of migration

The PCR product of ligation is not in high concentration (mostly with phusion polymerase) Following Gibson assembly was done by both fragments.

Gibson of cleaned up PCR products fragment 1 and fragment 2 in pSB1C3 treated by DpnI

By Manhaz

Gibson was performed with cleaned up PCR products fragment1 x fragment 2 x pSB1C3 treated by DpnI (plasmid) with the following protocol:

For each 20μl of reaction, mix the following reagents :

  • the 1-2 Gibson assembly product tube : 1,12µL of purified segment 1 and 0,56µL of purified segment 2 + 0,85µL of digested pSB1C3 + 7,47 µL of sterile water + 10µL of NEB Builder Hifi DNA Assembler Master Mix
  • the control tube : 0,85µL of digested pSB1C3 + 17,47 µL of sterile water

Gibson products were incubated 1h at 52°c.






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