Difference between revisions of "Team:Paris Saclay/Notebook/September/23"

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(→‎Lab work)
 
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= Friday 23<sup>rd</sup> September=
 
= Friday 23<sup>rd</sup> September=
−
==Lab work==
+
 
 
===Visualization===
 
===Visualization===
  
 +
====NanoDrop Measurements====
 +
''By Maxence, Manhaz & Coline''
  
−
==== Glycerol stocks of clones X, X, X and X containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)====
+
{| class="wikitable"
−
"By Maxence, Mahnaz & Coline"
+
!Sample
 +
!Concentration (ng/µL)
 +
|-
 +
|FKBP amplification product from the 8th September<div id="FKBP amplification product from the 8th September"></div>
 +
|231.55
 +
|-
 +
|gblock 2.2 amplification product from the 8th September<div id="gblock 2.2 amplification product from the 8th September"></div>
 +
|183.9
 +
|-
 +
|pSB1C3 treated by DpnI from the 15th September<div id="pSB1C3 treated by DpnI from the 15th September"></div>
 +
|59.68
 +
|-
 +
|PCR product containing FKBP - GFP 10 from the 22nd September<div id="PCR product containing FKBP - GFP 10 from the 22nd"></div>
 +
|38.19
 +
|-
 +
|}
 +
 
 +
As gblock 2.2 and FKBP were too concentrated, they were diluated at 1/10.
 +
 
 +
====Gibson of cleaned up PCR products FKBP from 8th September x gblock 2.2 from the 8th September x pSB1C3 treated by DpnI and Gibson of PCR product containing FKBP - GFP 10 from the 22nd September x pSB1C3 treated by DpnI ====
 +
''By Maxence, Manhaz & Coline''
 +
 
 +
Gibson was performed with cleaned up PCR products FKBP (insert 1) (obtained the 8th September) x gblock 2.2 (insert 2) (obtained the 8th September) x pSB1C3 treated by DpnI (plasmid) with the following protocol:
 +
 
 +
For each 20μl of reaction, mix the following reagents :
 +
* 1.74 µL of insert 1
 +
* 1.78 µL of insert 2
 +
* 1.67 µL of plasmid
 +
* 4.81 µL of water
 +
* 10 µL of buffer mix
 +
 
 +
Furthermore, Gibson was performed with cleaned up PCR containing FKBP - GFP 10 (insert) x pSB1C3 treated by DpnI (plasmid) with the following protocol:
 +
 
 +
For each 20μl of reaction, mix the following reagents :
 +
* 1.89 µL of insert
 +
* 1.67 µL of plasmid
 +
* 6.44 µL of water
 +
* 10 µL of buffer mix
 +
 
 +
Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). The PCR was performed as follow : 1 hour at 50°C.
 +
 
 +
====Transformation of DH5a cells with FKBP - GFP 10 in pSB1C3 (pPS16_018) obtained by Gibson====
 +
''By Maxence & Mahnaz''
 +
 
 +
Dh5a cells were transformed with pSB1C3 containing FKBP - GFP 10 (pPS16_018), or controls (no buffer mix and plasmid alone) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]].
 +
 
 +
==== Glycerol stocks of clones 2, 7 and 8 containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)====
 +
''By Maxence, Mahnaz & Coline''
  
 
The glycerol stock of the bacteria with the following plasmids were made.
 
The glycerol stock of the bacteria with the following plasmids were made.
−
*pPS16_022 (GFP 1.9) clone X
+
*pPS16_017 (dCas9 ST - GFP 11) clone 2
−
*pPS16_022 (GFP 1.9) clone X
+
*pPS16_017 (dCas9 ST - GFP 11) clone 7
−
*pPS16_022 (GFP 1.9) clone X
+
*pPS16_017 (dCas9 ST - GFP 11) clone 8
−
*pPS16_022 (GFP 1.9) clone X
+
  
 
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
 
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
  
−
====Plasmids extraction of clones X, X, X and X containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)====
+
====Plasmids extraction of clones 2, 7 and 8 containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)====
−
"By Maxence, Mahnaz & Coline"
+
''By Maxence, Mahnaz & Coline''
  
 
The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit:
 
The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit:
−
*pPS16_022 (GFP 1.9) clone X
+
*pPS16_017 (dCas9 ST - GFP 11) clone 2
−
*pPS16_022 (GFP 1.9) clone X
+
*pPS16_017 (dCas9 ST - GFP 11) clone 7
−
*pPS16_022 (GFP 1.9) clone X
+
*pPS16_017 (dCas9 ST - GFP 11) clone 8
−
*pPS16_022 (GFP 1.9) clone X
+
  
−
====Digestion of plasmids extracted from clones X, X, X and X containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)====
+
==== Glycerol stocks of clones 3, 4, 7 and 8 containing GFP 1.9 in pSB1C3 (pPS16_020)====
−
"By Maxence, Mahnaz & Coline"
+
''By Maxence, Mahnaz & Coline''
  
−
To assess if DH5a cells were transformed with the good plasmid, plasmid digestion was run with XbaI as following:
+
The glycerol stock of the bacteria with the following plasmids were made.
 +
*pPS16_020 (GFP 1.9) clone 3
 +
*pPS16_020 (GFP 1.9) clone 4
 +
*pPS16_020 (GFP 1.9) clone 7
 +
*pPS16_020 (GFP 1.9) clone 8
  
−
* 1 µL of extracted plasmid
+
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
−
* 1 µL of buffer
+
−
* 1 µL of restriction enzyme XbaI
+
−
* 7 µL of water
+
  
−
A control was done with 1 µL of non-digested plasmid.
+
====Plasmids extraction of clones 3, 4, 7 and 8 containing GFP 1.9 in pSB1C3 (pPS16_020)====
 +
''By Maxence, Mahnaz & Coline''
  
−
GEL
+
The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit:
 +
*pPS16_020 (GFP 1.9) clone 3
 +
*pPS16_020 (GFP 1.9) clone 4
 +
*pPS16_020 (GFP 1.9) clone 7
 +
*pPS16_020 (GFP 1.9) clone 8
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 17:08, 9 October 2016

Friday 23rd September

Visualization

NanoDrop Measurements

By Maxence, Manhaz & Coline

Sample Concentration (ng/µL)
FKBP amplification product from the 8th September
 231.55
gblock 2.2 amplification product from the 8th September
 183.9
pSB1C3 treated by DpnI from the 15th September
 59.68
PCR product containing FKBP - GFP 10 from the 22nd September
 38.19

As gblock 2.2 and FKBP were too concentrated, they were diluated at 1/10.

Gibson of cleaned up PCR products FKBP from 8th September x gblock 2.2 from the 8th September x pSB1C3 treated by DpnI and Gibson of PCR product containing FKBP - GFP 10 from the 22nd September x pSB1C3 treated by DpnI

By Maxence, Manhaz & Coline

Gibson was performed with cleaned up PCR products FKBP (insert 1) (obtained the 8th September) x gblock 2.2 (insert 2) (obtained the 8th September) x pSB1C3 treated by DpnI (plasmid) with the following protocol:

For each 20μl of reaction, mix the following reagents :

  • 1.74 µL of insert 1
  • 1.78 µL of insert 2
  • 1.67 µL of plasmid
  • 4.81 µL of water
  • 10 µL of buffer mix

Furthermore, Gibson was performed with cleaned up PCR containing FKBP - GFP 10 (insert) x pSB1C3 treated by DpnI (plasmid) with the following protocol:

For each 20μl of reaction, mix the following reagents :

  • 1.89 µL of insert
  • 1.67 µL of plasmid
  • 6.44 µL of water
  • 10 µL of buffer mix

Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). The PCR was performed as follow : 1 hour at 50°C.

Transformation of DH5a cells with FKBP - GFP 10 in pSB1C3 (pPS16_018) obtained by Gibson

By Maxence & Mahnaz

Dh5a cells were transformed with pSB1C3 containing FKBP - GFP 10 (pPS16_018), or controls (no buffer mix and plasmid alone) using the usual protocol.

Glycerol stocks of clones 2, 7 and 8 containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)

By Maxence, Mahnaz & Coline

The glycerol stock of the bacteria with the following plasmids were made.

  • pPS16_017 (dCas9 ST - GFP 11) clone 2
  • pPS16_017 (dCas9 ST - GFP 11) clone 7
  • pPS16_017 (dCas9 ST - GFP 11) clone 8

500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.

Plasmids extraction of clones 2, 7 and 8 containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)

By Maxence, Mahnaz & Coline

The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:

  • pPS16_017 (dCas9 ST - GFP 11) clone 2
  • pPS16_017 (dCas9 ST - GFP 11) clone 7
  • pPS16_017 (dCas9 ST - GFP 11) clone 8

Glycerol stocks of clones 3, 4, 7 and 8 containing GFP 1.9 in pSB1C3 (pPS16_020)

By Maxence, Mahnaz & Coline

The glycerol stock of the bacteria with the following plasmids were made.

  • pPS16_020 (GFP 1.9) clone 3
  • pPS16_020 (GFP 1.9) clone 4
  • pPS16_020 (GFP 1.9) clone 7
  • pPS16_020 (GFP 1.9) clone 8

500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.

Plasmids extraction of clones 3, 4, 7 and 8 containing GFP 1.9 in pSB1C3 (pPS16_020)

By Maxence, Mahnaz & Coline

The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:

  • pPS16_020 (GFP 1.9) clone 3
  • pPS16_020 (GFP 1.9) clone 4
  • pPS16_020 (GFP 1.9) clone 7
  • pPS16_020 (GFP 1.9) clone 8





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