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

(→‎PCR of cleaned up Ligation products (fragment 1 and fragment 2))
(→‎Wednesday 14th September)
Line 28: Line 28:
 
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 (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) ====
+
====PCR of gblock 1.1 - 1.2 (fragment 1) and gblock 2.1 - 2.2 (fragment 2) Ligation products====
 
''By Maxence''
 
''By Maxence''
  
Line 84: Line 84:
 
|iPS 123 and iPS84
 
|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''
 +
 +
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
 +
|-
 +
|}
 +
 +
GEL bizare
 +
 +
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.
 +
 +
GEL
  
 
====PCR of gblock detection, gblock ST sgRNA, gblock NM sgRNA, gblock spacer and plasmid PZA11 ====
 
====PCR of gblock detection, gblock ST sgRNA, gblock NM sgRNA, gblock spacer and plasmid PZA11 ====
Line 154: Line 182:
 
!20sec
 
!20sec
 
!30sec
 
!30sec
 +
|}
 +
 +
====PCR Clean-up of PCR products ====
 +
''By Maxence''
 +
 +
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]]. For each amplification, only PCR products from plasmids were cleaned in order to reduce the risks of mutations. For GFP 1.9 amplification products, the 4 samples were pooled before clean up.
 +
 +
====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
 +
|}
 +
 +
GEL ?
 +
 +
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).
 +
 +
GEL
 +
 +
====PCR of pSB1C3 from dCas9 ST - GFP 11 clone 8 ====
 +
''By Maxence''
 +
 +
In order to obtain FKBP - GFP 10 in pSB1C3 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 clone 8
 +
|-
 +
|Primers
 +
|iPS148 and iPS172
 +
|-
 
|}
 
|}

Revision as of 16:07, 14 September 2016

Wednesday 14th September

Lab work

Visualization

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

"By Maxence"

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

  • pPS16_009 (GFP 1.9) clone 2
  • pPS16_009 (GFP 1.9) clone 7
  • pPS16_014 (GFP 1.9) clone 8
  • pPS16_014 (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

"By Maxence"

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

  • pPS16_009 (GFP 1.9) clone 2
  • pPS16_009 (GFP 1.9) clone 7
  • pPS16_014 (GFP 1.9) clone 8
  • pPS16_014 (GFP 1.9) clone 12

Samples preparation for sequencing

By Maxence

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.

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

By Maxence

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

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

GEL bizare

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.

GEL

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

By Maxence

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.


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 gblock spacer in X gblock ST sgRNA in pUC19 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

PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol. For each amplification, only PCR products from plasmids were cleaned in order to reduce the risks of mutations. For GFP 1.9 amplification products, the 4 samples were pooled before clean up.

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

GEL ?

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).

GEL

PCR of pSB1C3 from dCas9 ST - GFP 11 clone 8

By Maxence

In order to obtain FKBP - GFP 10 in pSB1C3 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 clone 8
Primers iPS148 and iPS172