Difference between revisions of "Team:Paris Saclay/Notebook/August/9"

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= Tuesday 9<sup>th</sup> August=
 
= Tuesday 9<sup>th</sup> August=
−
==Lab work==
+
 
 
===Visualization===
 
===Visualization===
 
====Heat shock transformation====
 
====Heat shock transformation====
 
''By Charlène and Terrence''
 
''By Charlène and Terrence''
  
−
For 1.2, 4.2, SgNm, SgSt1, Detection, FRB and FKBP gBlocks, the results of the sequencing were not as expected.So, we transformed  
+
For 1.2, 4.2, SgNm, SgSt1, Detection, FRB and FKBP gBlocks, the sequençing results were not as expected. So, we transformed  
 
pPS16_002, pPS16_008, pPS16_010, pPS16_011, pPS16_012, pPS16_013 and pPS16_014 in DH5a. We followed the usual [[Team:Paris_Saclay/Experiments#Heat_shock_competent_cells|protocol]].
 
pPS16_002, pPS16_008, pPS16_010, pPS16_011, pPS16_012, pPS16_013 and pPS16_014 in DH5a. We followed the usual [[Team:Paris_Saclay/Experiments#Heat_shock_competent_cells|protocol]].
 
50µL of cells were streaked on LB + Amp (50µg/mL) + IPTG + xGal.
 
50µL of cells were streaked on LB + Amp (50µg/mL) + IPTG + xGal.
  
−
==== Phusion PCR on pPS16_006 and pPS16_007 ====
+
====Extraction of pPS16_001, pPS16_005, pPS16_006, pPS16_007 and pPS16_009====
 
''By Caroline''
 
''By Caroline''
  
−
The PCR was carried out following the usual [[Team:Paris_Saclay/Experiments#phusion|protocol]] adapted to 50µL and with a TM at 72°C. The specific [[Team:Paris_Saclay/Experiments#primers|primers]] for each parts were using. The products were put to migrated on a 0.8%agarose gel with BET.
+
Plasmids pPS16_001 (1.1), pPS16_005(3.1), pPS16_006(3.2), pPS16_007(4.1) and pPS16_009 (GFP) were extracted folllowing [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtractio|this protocol]].
 +
 
 +
 
 +
[[File:T--Paris_Saclay--160809_gel_extraction.JPG|400px|thumb|right|Result of the extraction]]
 +
 
 +
==== Phusion PCR on pPS16_001, pPS16_005, pPS16_006, pPS16_007 and pPS16_009====
 +
''By Caroline''
 +
 
 +
The PCR was carried out following the usual [[Team:Paris_Saclay/Experiments#phusion|protocol]] adapted to 50µL and with a Tm at 72°C. The specific [[Team:Paris_Saclay/Experiments#primers|primers]] for each parts were used. The products were put for migration on a 0.8%agarose gel with BET.
 +
Gel 1
 +
 
 +
PCR was made again to increase PCR products quantities.
 +
gel2
 +
 
 +
 
 +
pPS16_001 (1.1), pPS16_005 (3.1), pPS16_006 (3.2), pPS16_007 (4.1) and pPS16_009 (GFP).
 +
[[File:T--Paris Saclay--160809 gel PCR Phusion.JPG|400px|thumb|right|Result of the PCR]]
  
 
====pUC19 digestion with HincII enzyme====
 
====pUC19 digestion with HincII enzyme====
Line 21: Line 37:
 
5 µL of pUC19 plasmids were digested with 5µL of tango buffer 10X, 38µL of sterile water, and 1 µL of HincII enzyme. The mix was incubated at 37°C for 1 hour. After incubation, 1µL of HincII enzyme was added again, and the mix was incubated 1 hour again. Digestion products were migrated on a gel on [[Team:Paris_Saclay/Notebook/August/10#August_10|August 10]].
 
5 µL of pUC19 plasmids were digested with 5µL of tango buffer 10X, 38µL of sterile water, and 1 µL of HincII enzyme. The mix was incubated at 37°C for 1 hour. After incubation, 1µL of HincII enzyme was added again, and the mix was incubated 1 hour again. Digestion products were migrated on a gel on [[Team:Paris_Saclay/Notebook/August/10#August_10|August 10]].
  
−
===pUC19 ligation with gBlocks ATG linker RFB, detection, St sgRNA, ATG linker FKBP, Nm sgRNA, 1.2 and 4.2===
+
====pUC19 ligation with gBlocks ATG linker RFB, detection, St sgRNA, ATG linker FKBP, Nm sgRNA, 1.2 and 4.2====
 
''By Alice''
 
''By Alice''
 +
 +
Ligations of gBlocks with pUC19 were performed following [[Team:Paris_Saclay/Experiments#Ligation|this protocol]]. The final mix was incubated at 4°C overnight.
 +
Two control conditions were tested:
 +
#1µL of linearized pUC19 + 9 µL of sterile water
 +
#1µL of linearized pUC19 + 1 µL of ligase + 1µL of ligase buffer + 7µL of sterile water
 +
 +
With this ligation, we hope to get the plasmids below:
 +
{| class="wikitable"
 +
|-
 +
!'''gBlock'''
 +
!ATG linker RFB
 +
!detection
 +
!St sgRNA
 +
!ATG linker FKBP
 +
!Nm sgRNA
 +
|-
 +
!'''Plasmid name'''
 +
|pPS16_010<div id="pPS16_010"></div>
 +
|pPS16_011<div id="pPS16_011"></div>
 +
|pPS16_012<div id="pPS16_012"></div>
 +
|pPS16_013<div id="pPS16_013"></div>
 +
|pPS16_014<div id="pPS16_014"></div>
 +
|}
 +
 +
===Bringing DNA closer===
 +
====Preculture of DH5a cells transformed with Sp and Td dCas9====
 +
''By Léa''
 +
 +
200mL of liquid LB medium (spectinomycin 50µg/mL) were inoculated with DH5a cells transformed with Sp or Td dCas9 from glycerol stock, in order to extract the plasmids.
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 16:27, 9 October 2016

Tuesday 9th August

Visualization

Heat shock transformation

By Charlène and Terrence

For 1.2, 4.2, SgNm, SgSt1, Detection, FRB and FKBP gBlocks, the sequençing results were not as expected. So, we transformed pPS16_002, pPS16_008, pPS16_010, pPS16_011, pPS16_012, pPS16_013 and pPS16_014 in DH5a. We followed the usual protocol. 50µL of cells were streaked on LB + Amp (50µg/mL) + IPTG + xGal.

Extraction of pPS16_001, pPS16_005, pPS16_006, pPS16_007 and pPS16_009

By Caroline

Plasmids pPS16_001 (1.1), pPS16_005(3.1), pPS16_006(3.2), pPS16_007(4.1) and pPS16_009 (GFP) were extracted folllowing this protocol.


Result of the extraction

Phusion PCR on pPS16_001, pPS16_005, pPS16_006, pPS16_007 and pPS16_009

By Caroline

The PCR was carried out following the usual protocol adapted to 50µL and with a Tm at 72°C. The specific primers for each parts were used. The products were put for migration on a 0.8%agarose gel with BET. Gel 1

PCR was made again to increase PCR products quantities. gel2


pPS16_001 (1.1), pPS16_005 (3.1), pPS16_006 (3.2), pPS16_007 (4.1) and pPS16_009 (GFP).

Result of the PCR

pUC19 digestion with HincII enzyme

By Alice

5 µL of pUC19 plasmids were digested with 5µL of tango buffer 10X, 38µL of sterile water, and 1 µL of HincII enzyme. The mix was incubated at 37°C for 1 hour. After incubation, 1µL of HincII enzyme was added again, and the mix was incubated 1 hour again. Digestion products were migrated on a gel on August 10.

pUC19 ligation with gBlocks ATG linker RFB, detection, St sgRNA, ATG linker FKBP, Nm sgRNA, 1.2 and 4.2

By Alice

Ligations of gBlocks with pUC19 were performed following this protocol. The final mix was incubated at 4°C overnight. Two control conditions were tested:

  1. 1µL of linearized pUC19 + 9 µL of sterile water
  2. 1µL of linearized pUC19 + 1 µL of ligase + 1µL of ligase buffer + 7µL of sterile water

With this ligation, we hope to get the plasmids below:

gBlock ATG linker RFB detection St sgRNA ATG linker FKBP Nm sgRNA
Plasmid name pPS16_010
 pPS16_011
 pPS16_012
 pPS16_013
 pPS16_014

Bringing DNA closer

Preculture of DH5a cells transformed with Sp and Td dCas9

By Léa

200mL of liquid LB medium (spectinomycin 50µg/mL) were inoculated with DH5a cells transformed with Sp or Td dCas9 from glycerol stock, in order to extract the plasmids.





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