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

(Gel Electrophoresis of Gibson Assembly(fragment 3-4) and GFP 1-9 cloned in PSB1C3)
(Gel Electrophoresis of Gibson Assembly(fragment 3-4) and GFP 1-9 cloned in PSB1C3)
Line 11: Line 11:
 
[[File:T--Paris_Saclay--20160824_PCR_transfo_GFP_PSB1C3.jpg|400px|thumb|right|Colony PCR of 12 colonies containing the GFP 1-9.]]
 
[[File:T--Paris_Saclay--20160824_PCR_transfo_GFP_PSB1C3.jpg|400px|thumb|right|Colony PCR of 12 colonies containing the GFP 1-9.]]
  
  <br>
+
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
   
 
- Clones 2, 4, 7 and 11 are as expected.
 
- Clones 2, 4, 7 and 11 are as expected.
  

Revision as of 16:07, 25 August 2016

Wednesday 24th August

Lab work

Visualization

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

By Terrence

Colony PCR of 12 colonies containing the Gibson Assembly's 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 :

size (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

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.

PCR Q5 on plasmid

By Léa & Manhaz