Difference between revisions of "Team:Paris Saclay/Notebook/August/25"

(→‎Digestion of PSB1C3 containing the fragment 3 + 4 assembly and PSB1C3 with GFP1-9's insert.)
(→‎Visualization)
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====Q5 PCR on products of 1.1 and 1.2, and of 2.1 and 2.2 gBlocks ligation====
 +
''By Mathilde''
 +
 +
Q5 PCR was performed directly on gBlocks to amplify them following the protocol:
 +
*Prepare enough PCR master mix for two sets of triplicats analyzed plus one extra. For each 50μl of reaction, mix the following reagents :
 +
• 1µL of ligation product
 +
• 1µ of dNTPs (10mM)
 +
• 1µL of each primer mix (10µM)
 +
• 10µL of q5 buffer
 +
• 0,25µL of Q5 high fidelity polymerase
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• 35,75µL of nuclease free water
 +
 +
*Mix gently and aliquot 50μl of the mix into the PCR tubes on ice.
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*Perform PCR as follow:
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{| class="wikitable"
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|-
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!Step
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!Temperature
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!Time
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|-
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|Initial denaturation
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|98°C
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|30sec
 +
|-
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|rowspan="3"|30 cycles
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|98°C
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|5sec
 +
|-
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|72°c
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|30sec
 +
|-
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|72°C
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|1min
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|-
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|Final Extension
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|72°C
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|2min
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|-
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|Hold
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|4°C
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|$\infty$
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|}
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[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 +
 +
{| class="wikitable"
 +
|-
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!gBlocks
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!1.1 and 1.2 gBlocks ligation
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!2.1 and 2.2 gBlocks ligation
 +
|-
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|Primers
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|iPS83 and iPS122
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|iPS184 and iPS123
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|}
 +
 +
After amplification, 3 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.
 +
 +
PCR products expected were :
 +
 +
{| class="wikitable"
 +
|-
 +
!gBlocks
 +
!expected band size (bp)
 +
|-
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|1.1 and 1.2 gBlocks ligation
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|960
 +
|-
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|4.1 and 4.2 gBlocks ligation
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|1994
 +
|}
 +
 +
[[File:T--Paris_Saclay--20160817_PCR_gblocks_ligation.jpeg|500px|thumb|right|Migration of gBlocks and ligation]]
  
  

Revision as of 13:40, 25 August 2016

Thursday 25th August

Lab work

Visualization

Extraction of plasmids containing sgRNA Nm, fragment 3 + 4 (from the HIfi assembly mix) , GFP1-9

By Terrence

The extraction was carried out following the usual protocol.

The plasmid PSB1C3 with the insert : - SgRNA Nm were extracted from the clone 5 - Fragment 3 + 4 were extracted from the clone 6 - GFP1-9 were extracted from the clone 4



Digestion of PSB1C3 containing the fragment 3 + 4 assembly and PSB1C3 with GFP1-9's insert.

By Terrence

Extracted plasmids were digest with XbaI and PstI, following usual protocol.


Result of the digestion


Q5 PCR on products of 1.1 and 1.2, and of 2.1 and 2.2 gBlocks ligation

By Mathilde

Q5 PCR was performed directly on gBlocks to amplify them following the protocol:

  • Prepare enough PCR master mix for two sets of triplicats analyzed plus one extra. For each 50μl of reaction, mix the following reagents :

• 1µL of ligation product • 1µ of dNTPs (10mM) • 1µL of each primer mix (10µM) • 10µL of q5 buffer • 0,25µL of Q5 high fidelity polymerase • 35,75µL of nuclease free water

  • Mix gently and aliquot 50μl 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 5sec
72°c 30sec
72°C 1min
Final Extension 72°C 2min
Hold 4°C $\infty$ 
Primers used were:
gBlocks 1.1 and 1.2 gBlocks ligation 2.1 and 2.2 gBlocks ligation
Primers iPS83 and iPS122 iPS184 and iPS123

After amplification, 3 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.

PCR products expected were :

gBlocks expected band size (bp)
1.1 and 1.2 gBlocks ligation 960
4.1 and 4.2 gBlocks ligation 1994
Migration of gBlocks and ligation







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