Difference between revisions of "Team:Paris Saclay/Notebook/August/17"

(→‎Wednesday 17th August)
(→‎Q5 PCR on gblocks 1.2, NM_Sg_RNA, FRB and FKBP)
Line 31: Line 31:
 
Salicylate (main solution at 10mM) was added to obtain the following concentrations : 0, 0.03mM and 1mM.
 
Salicylate (main solution at 10mM) was added to obtain the following concentrations : 0, 0.03mM and 1mM.
  
−
====Q5 PCR on gblocks 1.2, NM_Sg_RNA, FRB and FKBP====
+
====Q5 PCR on gBlocks (1.2, NM_Sg_RNA, FRB and FKBP) and products of 4.1 and 4.2 gBlocks ligation====
 
''By Alice''
 
''By Alice''
  
−
Q5 PCR was performed directly on gBlocks to try to amplified it.  
+
Q5 PCR was performed directly on gBlocks to try to amplified it following [[Team:Paris_Saclay/Experiments#Q5PCR|this protocol]].
 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 +
 
 +
{| class="wikitable"
 +
|-
 +
!gBlocks
 +
!1.2
 +
!NM_Sg_RNA
 +
!FRB
 +
!FKBP
 +
!4.1 and 4.2 gBlocks ligation
 +
|-
 +
|Primers
 +
|iPS121 ans iPS122
 +
|iPS133 ans iPS83
 +
|iPS149 ans iPS150
 +
|iPS145 ans iPS146
 +
|iPS129 ans iPS84
 +
|}
 +
 
 +
Annealing temperature was 70°C.  After amplification, 1 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)
 +
|-
 +
|1.2
 +
|069
 +
|-
 +
|NM_Sg_RNA
 +
|362
 +
|-
 +
|FRB
 +
|473
 +
|-
 +
|FKBP
 +
|419
 +
|-
 +
|4.1 and 4.2 gBlocks ligation
 +
|1994
 +
|}
  
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Revision as of 15:36, 17 August 2016

Wednesday 17th August

Lab work

Visualization

Extraction of pPS16003, pPS16004 and GFP1-9_PSB1C3 (clones 2 and 3)

By Charlène

Clones 2 and 3 of GFP1-9_PSB1C3, and pPS16003, pPS16004 were extracted with the Plasmid MiniPrep kit.

The extracts were put to migrated on a 0.8%agarose gel with BET.

Purification of gBlocks 2, 3 and 4

By Terrence

The purification was carried out following the usual protocol.


Cloning with pJET the gBlock 1.2, FRB, FKBP and SgNM

By Léa and Naiane

The cloning was carried out using a new protocol which uses pJET as cloning vector.

A heat shock transformation was made on the cloning samples using the following protocol


Culture of BL21

By Charlène

3 clones from each transformation condition were grown in 500µL of LB, streptomycin (50µg/mL) and chloramphenicol (30µg/mL). Then each solution was split into 3x150µL and 350µL of mix of LB + streptomycin (50µg/mL) + chloramphenicol (30µg/mL) were added to each culture. Salicylate (main solution at 10mM) was added to obtain the following concentrations : 0, 0.03mM and 1mM.

Q5 PCR on gBlocks (1.2, NM_Sg_RNA, FRB and FKBP) and products of 4.1 and 4.2 gBlocks ligation

By Alice

Q5 PCR was performed directly on gBlocks to try to amplified it following this protocol. Primers used were:

gBlocks 1.2 NM_Sg_RNA FRB FKBP 4.1 and 4.2 gBlocks ligation
Primers iPS121 ans iPS122 iPS133 ans iPS83 iPS149 ans iPS150 iPS145 ans iPS146 iPS129 ans iPS84

Annealing temperature was 70°C. After amplification, 1 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)
1.2 069
NM_Sg_RNA 362
FRB 473
FKBP 419
4.1 and 4.2 gBlocks ligation 1994






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.