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− [[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|right|The three different samples of segment 1 and 2 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]]
+ [[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|right|Result of the migration]]
+
+ 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====
====pPS16_003 and pPS16_004 Ligation====
Revision as of 11:15, 26 August 2016
Thursday 25th August Lab work Visualization By Terrence
The extraction was carried out following the usual protocol .
The plasmid PSB1C3 with the insert :
- SgRNA Nm were extracted from the clone 5
- Fragment 3 + 4 were extracted from the clone 6
- GFP1-9 were extracted from the clone 4
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 usual protocol .
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:
Step
Temperature
Time
Initial denaturation
98°C
30sec
30 cycles
98°C
5sec
72°c
30sec
72°C
1min
Final Extension
72°C
2min
Hold
4°C
$\infty$
Primers used were:
gBlocks
1.1 and 1.2 gBlocks ligation
2.1 and 2.2 gBlocks ligation
Primers
iPS83 and iPS122
iPS184 and iPS123
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.
PCR products expected were :
gBlocks
expected band size (bp)
1.1 and 1.2 gBlocks ligation
1920
2.1 and 2.2 gBlocks ligation
1831
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
We can't see anything, the only visibles strands are the priers sequence, The result is not satisfying
