Difference between revisions of "Team:Paris Saclay/Notebook/August/25"

Revision as of 14:56, 20 September 2016

Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB

Mahnaz

Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 12 clones containing sg-Nm and 12 clones containing FRB were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 50 µg/mL Amp and liquid cultures (LB + 50 µg/mL Amp) 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)
0.5 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
25 cycles	94°C	30 sec
	Tm	30 sec
	72°C	t
Final Extension	72°C	7min
Hold	4°C	$\infinity\$

Primers used were:

Matrix	plasmid pJET coding sg-Nm	plasmid pJET coding FRB
Primers	pJET R and pJET F
Tm	60.0C
t	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 :

PCR products	Expected band size (bp)
dCas9 NM - GFP 10 - pSB1C3	3688

Result of the migration

GEL GEL GEL

We can't see anything, the only visibles strands are the priers sequence, The result is not satisfying

@@ Line 1: / Line 1: @@
-{{Team:Paris_Saclay/notebook_header}}
+====Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB====
-= Thursday 25<sup>th</sup> August=
+''Mahnaz''
-==Lab work==
-===Visualization===
-====Extraction of plasmids containing sgRNA Nm, fragment 3 + 4 (from the HIfi assembly mix) , GFP1-9====
-''By Terrence''
-The extraction was carried out following the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|usual protocol]].
+Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 12 clones containing sg-Nm and 12 clones containing FRB 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 + 50 µg/mL Amp and liquid cultures (LB + 50 µg/mL Amp) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.
-The plasmid PSB1C3  with the insert :
+For each clones contained in 20 μl water, 4.13 μL of the following mix were added :
-- SgRNA Nm were extracted from the  clone 5
+* 2.5 µL DreamTaq Buffer
-- Fragment 3 + 4 were extracted from the  clone 6
+* 0.5 µL of dNTPs (10mM)
-- GFP1-9 were extracted from the  clone 4
+* 0.5 µL of each primer mix (10µM)
+* 0.13 μl of DreamTaq Pol
+PCR was performed as follow:
-====Digestion of PSB1C3 containing the fragment 3 + 4 assembly and PSB1C3 with GFP1-9's insert.====
-''By Terrence''
-Extracted plasmids were digest with XbaI and PstI, following [[Team:Paris_Saclay/Experiments#PlasmidDigestion|usual protocol]].
-[[File:T--Paris_Saclay--20160825_digestion_gel.jpg|400px|thumb|right|Result of the digestion]]
-====Q5 PCR on products of 1.1 and 1.2, and of 2.1 and 2.2 gBlocks ligation====
-''By Mathilde''
-Q5 PCR was performed directly on gBlocks to amplify them following the protocol:
-Prepare enough PCR master mix for two sets of triplicats analyzed plus one extra. For each 50μl of reaction, mix the following reagents :
-* 1µL of ligation product
-* 1µ of dNTPs (10mM)
-* 1µL of each primer mix (10µM)
-* 10µL of q5 buffer
-* 0,25µL of Q5 high fidelity polymerase
-* 35,75µL of nuclease free water
-Mix gently and aliquot 50μl of the mix into the PCR tubes on ice.
-Perform PCR as follow:
 {| class="wikitable"
 |-
@@ Line 47: / Line 18: @@
 |-
 |Initial denaturation
-|98°C
+|95°C
-|30sec
+|3 min
 |-
-|rowspan="3"|30 cycles
+|rowspan="3"|25 cycles
-|98°C
+|94°C
-|5sec
+|30 sec
 |-
-|72°c
+|Tm
-|30sec
+|30 sec
 |-
 |72°C
-|1min
+|t
 |-
 |Final Extension
 |72°C
-|2min
+|7min
 |-
 |Hold
 |4°C
-|$\infty$
+|$\infinity\$
 |}
   [[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
@@ Line 74: / Line 44: @@
 {| class="wikitable"
 |-
-!gBlocks
+!Matrix
-!1.1 and 1.2 gBlocks ligation
+!plasmid pJET coding sg-Nm
-!2.1 and 2.2 gBlocks ligation
+!plasmid pJET coding FRB
 |-
 |Primers
-|iPS83 and iPS122
+|pJET R and pJET F
-|iPS184 and iPS123
+|-
+|Tm
+|60.0C
+|-
+|t
+|30 sec
 |}
-After amplification, 3 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.
+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 :
@@ Line 89: / Line 64: @@
 {| class="wikitable"
 |-
-!gBlocks
+!PCR products
-!expected band size (bp)
+!Expected band size (bp)
 |-
-|1.1 and 1.2 gBlocks ligation
+|dCas9 NM - GFP 10 - pSB1C3
-|1920
+|3688
-|-
-|2.1 and 2.2 gBlocks ligation
-|1831
 |}
 [[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]]
+GEL GEL GEL
-The three different samples of segment 1 (ligated pPS16_001 and pPS16_002) and segment 2  (ligated pPS16_001 and pPS16_002) PCR product seem to be at the expected size. But segment 2 is in insufficient quantity, so the segment 2 ligation and purification will be conducted again.
-====pPS16_003 and pPS16_004 Ligation====
-''By Mathilde''
-gBlocks pPS16_003 and pPS16_004 were ligated together as following :
-* 4µL of pPS16_00 PCR product from the 08/08/2016
-* 4µM of pPS16_004 PCR product from 08/08/2016
-* 1µL of Buffer T4 10X
-* 1µL of ligase T4 enzyme
-The ligation product was put at 4°c overnight.
-====PCR Colony  ====
-''By Mahnaz''
 [[File:T--Paris_Saclay--20160825_PCR_gel_Nm_Frb.jpg|400px|thumb|right|We can't see anything, the only visibles strands are the priers sequence, The result is not satisfying]]