Difference between revisions of "Team:Paris Saclay/Notebook/September/14"

(→‎PCR of cleaned up Ligation products (fragment 1 and fragment 2))
(→‎Lab work)
 
(33 intermediate revisions by 3 users not shown)
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 +
{{Team:Paris_Saclay/notebook_header}}
 +
 
= Wednesday 14<sup>th</sup> September=
 
= Wednesday 14<sup>th</sup> September=
−
==Lab work==
+
 
 
===Visualization===
 
===Visualization===
  
−
==== Glycerol stocks of clones 2, 7, 8 and 12 containing GFP 1.9 in pSB1C3 ====
+
==== Glycerol stocks of clones 2, 7, 8 and 12 containing GFP 1.9 in pSB1C3 (pPS16_020)====
−
"By Maxence"
+
"By Maxence & Mahnaz"
  
 
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_009 (GFP 1.9) clone 2
+
*pPS16_020 (GFP 1.9) clone 2
−
*pPS16_009 (GFP 1.9) clone 7
+
*pPS16_020 (GFP 1.9) clone 7
−
*pPS16_014 (GFP 1.9) clone 8
+
*pPS16_020 (GFP 1.9) clone 8
−
*pPS16_014 (GFP 1.9) clone 12
+
*pPS16_020 (GFP 1.9) clone 12
  
 
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 2, 7, 8 and 12 containing GFP 1.9 in pSB1C3====
+
====Plasmids extraction of clones 2, 7, 8 and 12 containing GFP 1.9 in pSB1C3 (pPS16_020)====
−
"By Maxence"
+
"By Maxence & Mahnaz"
  
 
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_009 (GFP 1.9) clone 2
+
*pPS16_020 (GFP 1.9) clone 2
−
*pPS16_009 (GFP 1.9) clone 7
+
*pPS16_020 (GFP 1.9) clone 7
−
*pPS16_014 (GFP 1.9) clone 8
+
*pPS16_020 (GFP 1.9) clone 8
−
*pPS16_014 (GFP 1.9) clone 12
+
*pPS16_020 (GFP 1.9) clone 12
  
 
====Samples preparation for sequencing====
 
====Samples preparation for sequencing====
−
''By Maxence''
+
''By Maxence & Mahnaz''
  
−
20 µL of plasmids pSB1C3 GFP 1.9 (clones 2, 7, 8 and 12) were sent to be sequenced. 20 µL of the primers iPS84 (5µM) and iPS140 (5µM) were sent for sequencing.
+
20 µL of plasmids pSB1C3 GFP 1.9 (pPS16_020) (clones 2, 7, 8 and 12) were sent to be sequenced. 20 µL of the primers iPS84 (5µM) and iPS140 (5µM) were sent for sequencing.
  
−
====PCR of cleaned up Ligation products (fragment 1 and fragment 2) ====
+
====NanoDrop Measurements====
−
''By Maxence''
+
''By Maxence & Mahnaz''
 +
 
 +
{| class="wikitable"
 +
!Sample
 +
!Concentration (ng/µL)
 +
|-
 +
|GFP 1.9 clone 2<div id="GFP 1.9 clone 2"></div>
 +
|24.84
 +
|-
 +
|GFP 1.9 clone 7<div id="GFP 1.9 clone 7"></div>
 +
|95.16
 +
|-
 +
|GFP 1.9 clone 8<div id="GFP 1.9 clone 8"></div>
 +
|145.6
 +
|-
 +
|GFP 1.9 clone 12<div id="GFP 1.9 clone 12"></div>
 +
|42.18
 +
|-
 +
|}
 +
 
 +
====PCR of gblock 1.1 - 1.2 (fragment 1) and gblock 2.1 - 2.2 (fragment 2) Ligation products====
 +
''By Maxence & Mahnaz''
  
 
With the results obtained the 12th and 13th September, a new amplification approach was tested: the Ligation products from the 12th were diluated at 1/10 and two buffer (HF and GC) were tested with others different PCR conditions. Furthermore, ligations products from the 13th September were also amplified.
 
With the results obtained the 12th and 13th September, a new amplification approach was tested: the Ligation products from the 12th were diluated at 1/10 and two buffer (HF and GC) were tested with others different PCR conditions. Furthermore, ligations products from the 13th September were also amplified.
Line 72: Line 95:
 
|}
 
|}
  
−
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
+
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
  
 
{| class="wikitable"
 
{| class="wikitable"
Line 85: Line 108:
 
|}
 
|}
  
−
====PCR of gblock detection, gblock ST sgRNA, gblock NM sgRNA, gblock spacer and plasmid PZA11 ====
+
====Gel of gblock 1.1 - 1.2 (fragment 1) and gblock 2.1 - 2.2 (fragment 2) Ligation products====
−
''By Maxence''
+
''By Maxence & Mahnaz''
  
−
In order to obtain pZA11 containing the desired sequences, gblocks were amplified in order to run Gibson. For that purpose, PCR was performed with the following protocol.
+
3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
 +
 
 +
PCR products expected were :
 +
 
 +
{| class="wikitable"
 +
|-
 +
!PCR products
 +
!Expected band size (bp)
 +
|-
 +
|Fragment 1
 +
|1920
 +
|-
 +
|Fragment 2
 +
|1831
 +
|-
 +
|}
 +
 
 +
[[File:T--Paris Saclay--Gel1112.png|400px|thumb|center|Result of the migration]]
 +
 
 +
The results were the same as previous ones.
 +
 
 +
Another strategy was to make a PCR directly of fragments 1 and 2 obtained previously even if these fragments were not working for Gibson. Fragments 1 and 2 from the 2nd September were amplified with the same protocol as mentioned above.
 +
 
 +
[[File:T--Paris Saclay--Gel1113.png|400px|thumb|center|Result of the migration]]
 +
 
 +
====PCR of gblock detection, gblock ST sgRNA, gblock NM sgRNA, gblock spacer and plasmid PZA11 ====
 +
''By Maxence & Mahnaz''
  
 +
In order to obtain pZA11 containing the desired sequences (pPS16_025), gblocks were amplified in order to run Gibson. For that purpose, PCR was performed with the following protocol.
  
 
For each 50μl of reaction, mix the following reagents :
 
For each 50μl of reaction, mix the following reagents :
Line 130: Line 180:
 
|}
 
|}
  
−
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
+
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
  
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
 
!Matrix
 
!Matrix
−
!gblock detection in pUC19
+
!gblock detection in pUC19 (pPS16_016)
−
!gblock spacer in X
+
!gblock spacer in pUC19 (pPS16_015)
−
!gblock ST sgRNA in pUC19
+
!gblock ST sgRNA in pUC19 (pPS16_012)
 
!gblock NM sgRNA in pJET
 
!gblock NM sgRNA in pJET
 
!extract of pzA11
 
!extract of pzA11
Line 155: Line 205:
 
!30sec
 
!30sec
 
|}
 
|}
 +
 +
====PCR Clean-up of PCR products ====
 +
''By Maxence & Mahnaz''
 +
 +
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]].
 +
 +
====Gel of cleaned up PCR products====
 +
''By Maxence''
 +
 +
After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
 +
 +
PCR products expected were :
 +
 +
{| class="wikitable"
 +
|-
 +
!PCR products
 +
!Expected band size (bp)
 +
|-
 +
|gblock detection
 +
|1020
 +
|-
 +
|gblock spacer
 +
|900
 +
|-
 +
|gblock ST sgRNA
 +
|310
 +
|-
 +
|gblock NM sgRNA
 +
|362
 +
|-
 +
|pZA11
 +
|2125
 +
|}
 +
 +
[[File:T--Paris Saclay--Gel1114.png|400px|thumb|center|Result of the migration]]
 +
 +
All PCR products were at the good size but there were no results obtained for NM sgRNA.
 +
 +
Another PCR was run in order to obtain PCR products from NM sgRNA, the good TM was used (63°C).
 +
 +
[[File:T--Paris Saclay--Gel1115.png|400px|thumb|center|Result of the migration]]
 +
 +
====Linearization of cleaned-up PCR product pZA11====
 +
''By Maxence & Mahnaz''
 +
 +
PCR product PZA11 has been linearized for further Gibson application by using DpnI treatment :
 +
* 30 µL of cleaned up PCR product GFP 11 - pSB1C3
 +
* 4 µL of fast digest buffer
 +
* 1 µL of DpnI
 +
* 5 µL of water
 +
 +
The mix was placed 10 minutes at 37°C and was cleaned by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]].
 +
 +
====PCR of pSB1C3 from dCas9 ST - GFP 11 (pPS16_017) clone 8 ====
 +
''By Maxence & Mahnaz''
 +
 +
In order to obtain FKBP - GFP 10 in pSB1C3 (pPS16_018) by Gibson, pSB1C3 must be amplified in order to run Gibson. For that purpose, PCR was performed with the following protocol.
 +
 +
For each 50μl of reaction, mix the following reagents :
 +
* 1 µL of matrix
 +
* 1 µL of dNTPs (10mM)
 +
* 2.5 µL of each primer mix (10µM)
 +
* 10 µL of buffer HF (5X)
 +
* 0,5 µL of Phusion polymerase
 +
* 32.5 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
 +
|-
 +
|67°C
 +
|30sec
 +
|-
 +
|72°C
 +
|1min
 +
|-
 +
|Final Extension
 +
|72°C
 +
|2min
 +
|-
 +
|Hold
 +
|4°C
 +
|$\infty$
 +
|}
 +
 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 +
 +
{| class="wikitable"
 +
|-
 +
!Matrix
 +
!dCas9 ST - GFP 11 (pPS16_017) clone 8
 +
|-
 +
|Primers
 +
|iPS148 and iPS172
 +
|-
 +
|}
 +
 +
====PCR Clean-up of PCR products pSB1C3 ====
 +
''By Maxence & Mahnaz''
 +
 +
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]].
 +
 +
====Gel of cleaned up PCR products pSB1C3====
 +
''By Maxence & Mahnaz''
 +
 +
After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
 +
 +
PCR products expected were :
 +
 +
{| class="wikitable"
 +
|-
 +
!PCR products
 +
!Expected band size (bp)
 +
|-
 +
|pSB1C3
 +
|2070
 +
|}
 +
 +
[[File:T--Paris Saclay--Gel1116.png|400px|thumb|center|Result of the migration]]
 +
 +
PCR products were at the good size.
 +
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 17:05, 9 October 2016

Wednesday 14th September

Visualization

Glycerol stocks of clones 2, 7, 8 and 12 containing GFP 1.9 in pSB1C3 (pPS16_020)

"By Maxence & Mahnaz"

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

  • pPS16_020 (GFP 1.9) clone 2
  • pPS16_020 (GFP 1.9) clone 7
  • pPS16_020 (GFP 1.9) clone 8
  • pPS16_020 (GFP 1.9) clone 12

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

Plasmids extraction of clones 2, 7, 8 and 12 containing GFP 1.9 in pSB1C3 (pPS16_020)

"By Maxence & Mahnaz"

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

  • pPS16_020 (GFP 1.9) clone 2
  • pPS16_020 (GFP 1.9) clone 7
  • pPS16_020 (GFP 1.9) clone 8
  • pPS16_020 (GFP 1.9) clone 12

Samples preparation for sequencing

By Maxence & Mahnaz

20 µL of plasmids pSB1C3 GFP 1.9 (pPS16_020) (clones 2, 7, 8 and 12) were sent to be sequenced. 20 µL of the primers iPS84 (5µM) and iPS140 (5µM) were sent for sequencing.

NanoDrop Measurements

By Maxence & Mahnaz

Sample Concentration (ng/µL)
GFP 1.9 clone 2
 24.84
GFP 1.9 clone 7
 95.16
GFP 1.9 clone 8
 145.6
GFP 1.9 clone 12
 42.18

PCR of gblock 1.1 - 1.2 (fragment 1) and gblock 2.1 - 2.2 (fragment 2) Ligation products

By Maxence & Mahnaz

With the results obtained the 12th and 13th September, a new amplification approach was tested: the Ligation products from the 12th were diluated at 1/10 and two buffer (HF and GC) were tested with others different PCR conditions. Furthermore, ligations products from the 13th September were also amplified.

For each 50μl of reaction, mix the following reagents :

  • 1 µL of matrix
  • 1 µL of dNTPs (10mM)
  • 2.5 µL of each primer mix (10µM)
  • 10 µL of buffer (5X)
  • 0,5 µL of Phusion polymerase
  • 32.5 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 95°C 30sec
25 cycles 95°C 30sec
50°C 30sec
72°C 30sec
Final Extension 72°C 5min
Hold 4°C $\infty$

Primers used were:

Matrix Products from Ligation of gblock 1.1 and gblock 1.2 (fragment 1) Products from Ligation of gblock 1.1 and gblock 1.2 (fragment 2)
Primers iPS140 and iPS122 iPS 123 and iPS84

Gel of gblock 1.1 - 1.2 (fragment 1) and gblock 2.1 - 2.2 (fragment 2) Ligation products

By Maxence & Mahnaz

3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

PCR products Expected band size (bp)
Fragment 1 1920
Fragment 2 1831
Result of the migration

The results were the same as previous ones.

Another strategy was to make a PCR directly of fragments 1 and 2 obtained previously even if these fragments were not working for Gibson. Fragments 1 and 2 from the 2nd September were amplified with the same protocol as mentioned above.

Result of the migration

PCR of gblock detection, gblock ST sgRNA, gblock NM sgRNA, gblock spacer and plasmid PZA11

By Maxence & Mahnaz

In order to obtain pZA11 containing the desired sequences (pPS16_025), gblocks were amplified in order to run Gibson. For that purpose, PCR was performed with the following protocol.

For each 50μl of reaction, mix the following reagents :

  • 1 µL of matrix
  • 1 µL of dNTPs (10mM)
  • 2.5 µL of each primer mix (10µM)
  • 10 µL of buffer HF (5X)
  • 0,5 µL of Phusion polymerase
  • 32.5 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 30sec
55°C 30sec
72°C t
Final Extension 72°C 5min
Hold 4°C $\infty$

Primers used were:

Matrix gblock detection in pUC19 (pPS16_016) gblock spacer in pUC19 (pPS16_015) gblock ST sgRNA in pUC19 (pPS16_012) gblock NM sgRNA in pJET extract of pzA11
Primers iPS153 and iPS154 iPS155 and iPS156 iPS157 and iPS158 iPS159 and iPS160 iPS161 and iPS162
t 20sec 20sec 20sec 20sec 30sec

PCR Clean-up of PCR products

By Maxence & Mahnaz

PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.

Gel of cleaned up PCR products

By Maxence

After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

PCR products Expected band size (bp)
gblock detection 1020
gblock spacer 900
gblock ST sgRNA 310
gblock NM sgRNA 362
pZA11 2125
Result of the migration

All PCR products were at the good size but there were no results obtained for NM sgRNA.

Another PCR was run in order to obtain PCR products from NM sgRNA, the good TM was used (63°C).

Result of the migration

Linearization of cleaned-up PCR product pZA11

By Maxence & Mahnaz

PCR product PZA11 has been linearized for further Gibson application by using DpnI treatment :

  • 30 µL of cleaned up PCR product GFP 11 - pSB1C3
  • 4 µL of fast digest buffer
  • 1 µL of DpnI
  • 5 µL of water

The mix was placed 10 minutes at 37°C and was cleaned by using the NucleoSpin Gel and PCR Clean-up kit protocol.

PCR of pSB1C3 from dCas9 ST - GFP 11 (pPS16_017) clone 8

By Maxence & Mahnaz

In order to obtain FKBP - GFP 10 in pSB1C3 (pPS16_018) by Gibson, pSB1C3 must be amplified in order to run Gibson. For that purpose, PCR was performed with the following protocol.

For each 50μl of reaction, mix the following reagents :

  • 1 µL of matrix
  • 1 µL of dNTPs (10mM)
  • 2.5 µL of each primer mix (10µM)
  • 10 µL of buffer HF (5X)
  • 0,5 µL of Phusion polymerase
  • 32.5 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
67°C 30sec
72°C 1min
Final Extension 72°C 2min
Hold 4°C $\infty$

Primers used were:

Matrix dCas9 ST - GFP 11 (pPS16_017) clone 8
Primers iPS148 and iPS172

PCR Clean-up of PCR products pSB1C3

By Maxence & Mahnaz

PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.

Gel of cleaned up PCR products pSB1C3

By Maxence & Mahnaz

After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

PCR products Expected band size (bp)
pSB1C3 2070
Result of the migration

PCR products were at the good size.





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