Difference between revisions of "Team:Paris Saclay/Notebook/September/7"

(→‎Wednesday 7th September)
(→‎Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3)
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Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 16 clones containing dCas9 NM - GFP 10 obtained by Q5 amplification and 16 clones containing dCas9 NM - GFP 10 obtained by Phusion amplification  were screened and used for the usual [[Team:Paris_Saclay/Experiments##Polymerase_chain_reaction|protocol]] of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.
 
Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 16 clones containing dCas9 NM - GFP 10 obtained by Q5 amplification and 16 clones containing dCas9 NM - GFP 10 obtained by Phusion amplification  were screened and used for the usual [[Team:Paris_Saclay/Experiments##Polymerase_chain_reaction|protocol]] of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.
  
−
For each 50μl of reaction, mix the following reagents :
+
For each clones contained in 20 μl water, 4.13 μL of the following mix were added :
−
* 1 µL of plasmid
+
* 2.5 µL DreamTaq Buffer
−
* 1 µL of dNTPs (10mM)
+
* 0.5 µL of dNTPs (10mM)
 
* 1 µL of each primer mix (10µM)
 
* 1 µL of each primer mix (10µM)
−
* 10 µL of Q5 buffer (5X)
+
* 0.13 μl of DreamTaq Pol
−
* 0,5 µL of Q5 high fidelity polymerase
+
−
* 35,5 µL of nuclease free water
+
  
−
Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice.
+
PCR was performed as follow:  
−
Perform PCR as follow:  
+
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
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|-
 
|-
 
|Initial denaturation
 
|Initial denaturation
−
|98°C
+
|95°C
−
|30sec
+
|3 min
 
|-
 
|-
 
|rowspan="3"|30 cycles
 
|rowspan="3"|30 cycles
−
|98°C
+
|95°C
−
|10sec
+
|30 sec
 
|-
 
|-
 
|Tm
 
|Tm
−
|20sec
+
|30 sec
 
|-
 
|-
 
|72°C
 
|72°C
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|Final Extension
 
|Final Extension
 
|72°C
 
|72°C
−
|2min
+
|7min
 
|-
 
|-
 
|Hold
 
|Hold
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|-
 
|-
 
!Matrix
 
!Matrix
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!dCas9 ST - GFP 11 in pSB1C3 clones 6 and 8
+
!Clones containing dCas9 NM - GFP 10 in pSB1C3
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!FRB in pJET clones 4 and 9
+
−
!GFP 1.9 in pUC19
+
 
|-
 
|-
 
|Primers
 
|Primers
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|iPS152 and iPS151
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|iPS83 and iPS84
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|iPS149 and iPS150
+
−
|iPS84 and iPS140
+
 
|-
 
|-
 
|Tm
 
|Tm
−
|57,5°C
+
|63.6°C
−
|56,7°C
+
−
|72°C
+
 
|-
 
|-
 
|t
 
|t
 
|1 min 30
 
|1 min 30
−
|20 sec
 
−
|30 sec
 
 
|}
 
|}
  
 +
After amplification, 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)
 +
|-
 +
|dCas9 NM - GFP 10 - pSB1C3
 +
|3688
 +
|}
  
 +
[[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]]
  
−
 
+
GEL GEL GEL
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+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
  
 
====Q5 PCR on dCas9 ST - GFP 11 in pSB1C3, FRB in pJET and GFP 1.9 in pUC19 ====
 
====Q5 PCR on dCas9 ST - GFP 11 in pSB1C3, FRB in pJET and GFP 1.9 in pUC19 ====

Revision as of 13:10, 13 September 2016

Wednesday 7th September

Lab work

Visualization

Gibson of cleaned up PCR products FRB from clone 9 x pSB1C3 - GFP 11 from clone 8

By Maxence

Gibson was performed on cleaned up PCR products FRB from clone 9 (insert) x pSB1C3 - GFP 11 from clone 8 (plasmid) with the following protocol:

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

  • 0.64 µL of insert
  • 3.68 µL of plasmid
  • 5.67 µL of water
  • 10 µL of buffer

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.

Clean-up of Gibson products

By Maxence

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

Transformation of DH5a cells with FRB - GFP 11 in pSB1C3 obtained by Gibson

By Maxence

Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11, or containing control (no mix added when Gibson performed) using the usual protocol.

Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3

Maxence

Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 16 clones containing dCas9 NM - GFP 10 obtained by Q5 amplification and 16 clones containing dCas9 NM - GFP 10 obtained by Phusion amplification were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.

For each clones contained in 20 μl water, 4.13 μL of the following mix were added :

  • 2.5 µL DreamTaq Buffer
  • 0.5 µL of dNTPs (10mM)
  • 1 µL of each primer mix (10µM)
  • 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step Temperature Time
Initial denaturation 95°C 3 min
30 cycles 95°C 30 sec
Tm 30 sec
72°C t
Final Extension 72°C 7min
Hold 4°C $\infinity\$ 
Primers used were:
Matrix Clones containing dCas9 NM - GFP 10 in pSB1C3
Primers iPS83 and iPS84
Tm 63.6°C
t 1 min 30

After amplification, 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)
dCas9 NM - GFP 10 - pSB1C3 3688
Result of the migration

GEL GEL GEL

Q5 PCR on dCas9 ST - GFP 11 in pSB1C3, FRB in pJET and GFP 1.9 in pUC19

By Maxence

Q5 PCR was performed on plasmids with the following protocol:

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

  • 1 µL of plasmid
  • 1 µL of dNTPs (10mM)
  • 1 µL of each primer mix (10µM)
  • 10 µL of Q5 buffer (5X)
  • 0,5 µL of Q5 high fidelity polymerase
  • 35,5 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
Tm 20sec
72°C t
Final Extension 72°C 2min
Hold 4°C $\infinity\$ 
Primers used were:
Matrix dCas9 ST - GFP 11 in pSB1C3 clones 6 and 8 FRB in pJET clones 4 and 9 GFP 1.9 in pUC19
Primers iPS152 and iPS151 iPS149 and iPS150 iPS84 and iPS140
Tm 57,5°C 56,7°C 72°C
t 1 min 30 20 sec 30 sec


NanoDrop Measurements

By Maxence

Sample Concentration (ng/µL)
PCR fragment GFP 11 clone 6
 187.23
PCR fragment GFP 11 clone 8
 156.85
PCR fragment FRB clone 4
 75.67
PCR fragment FRB clone 9
 246.41
PCR fragment GFP 1.9
 22.06

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)
GFP 11- pSB1C3 2500
FRB 374
GFP 1.9 862
Result of the migration

GEL GEL GEL