Difference between revisions of "Team:Paris Saclay/Notebook/August/24"

(→‎Plasmid DNA extraction (Midi Prep))
(→‎PCR Q5 on plasmid)
Line 88: Line 88:
 
====PCR Q5 on plasmid ====
 
====PCR Q5 on plasmid ====
 
''By Léa & Manhaz''
 
''By Léa & Manhaz''
 +
 +
Plasmid Pjet cotnains gblock 1.2 (confirmed by sequencing) was used as template to amplify the fragement 1.2.
 +
also we amplify the plasmids extracted from 2 clonies contrains the plasmid coding dCas Nm from adgene.
 +
 +
Q5 PCR was 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
 +
!gblock1.2 in pJET
 +
!plasmid adgene contain dCas9 Nm clones 1 and 2
 +
|-
 +
|Primers
 +
|iPS121 and iPS122
 +
|iPS121 and iPS122
 +
|-
 +
|Tm
 +
|72°C
 +
|72°C
 +
|-
 +
|t
 +
|30 sec
 +
|30 sec
 +
|}
 +
 +
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Revision as of 13:19, 20 September 2016

Wednesday 24th August

Lab work

Visualization

Gel Electrophoresis of Gibson Assembly's products(fragment 3-4) and GFP 1-9 cloned in PSB1C3

By Terrence

Colony PCR of 12 colonies transformed with Gibson Assembly products (fragment 3-4).
Colony PCR of 12 colonies containing the GFP 1-9.
- Clones 2, 4, 7 and 11 are as expected.

pPS16_002 (1.2) clones 8 and 14 and pPS16_014(SgRna Nm) clone 12 Extraction

By Terrence

The extraction was carried out following the usual protocol.


Result of the extraction


Nano drop :

concentration (ng/µL) 260/230 260/280
1.2.14 47.81 1.64 1.84
1.2.8 834.5 2.40 1.99
SgRna Nm 12 124.21 2.00 1.89

Transformation of gblockFRB in pJET and gblock Sg_RNA_NM in pJET

By Terrence


First we made a ligation with

Component Volume (µL)
Reaction Buffer 10
PCR Product (FRB or Nm) 1
pJET1.2/blunt 1
water, nuclease free 7
T4 DNA Ligase 1

Then we transformed 2µL of ligation product into 50 µL of DH5a competent cells, following the usual protocol.

Transformation products were plated with three different volume : 50µL , 150µl and 350µL. 350µL only for the control.

Plasmid DNA extraction (Midi Prep)

By Léa & Manhaz

We have decided to use the plasmid coding dCas9 Nm to amplify the fragement corresponding g-block 1.2. 200 mL of transformed DH5a cells with Adgene dCas9 Nm plasmids (clone 1 and 2) cultures were extracted usind a Midi Prep kit. It was resuspended into 50µL of sterile water.

The plasmid then reserved in -20.

PCR Q5 on plasmid

By Léa & Manhaz

Plasmid Pjet cotnains gblock 1.2 (confirmed by sequencing) was used as template to amplify the fragement 1.2. also we amplify the plasmids extracted from 2 clonies contrains the plasmid coding dCas Nm from adgene.

Q5 PCR was 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 gblock1.2 in pJET plasmid adgene contain dCas9 Nm clones 1 and 2
Primers iPS121 and iPS122 iPS121 and iPS122
Tm 72°C 72°C
t 30 sec 30 sec






Université Paris Sud
91405 Orsay, France
Copyright © 2016 iGEM Paris Saclay. All rights reserved All inspirations encouraged.
*This wiki was conceived during long nights of work and a great coffee experience in the beautiful Orsay, France.