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Line 4: ===Visualization===
===Visualization===
+
+ ====pPS16_014 (SgRNA Nm) clone 5, Gibson Assembly's Product (Fragment3-4) clone 6, and pPS16_009 clone 4 extraction====
+ ''By Terrence''
+
+ The extraction was carried out following the [[Team:Paris_Saclay/Experiments#DNA_extraction_using_the_Invitrogen_ChargeSwitch.C2.AE-Pro_Plasmid_Miniprep_Kit|usual protocol]]
====Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB====
====Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB====
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Line 132: * 1µL of T4 Ligase
* 1µL of T4 Ligase
The solution is put in incubation at 4°c over night.
The solution is put in incubation at 4°c over night.
−
{{Team:Paris_Saclay/notebook_footer}}
{{Team:Paris_Saclay/notebook_footer}}
Revision as of 11:55, 17 October 2016
Thursday 25th August Visualization pPS16_014 (SgRNA Nm) clone 5, Gibson Assembly's Product (Fragment3-4) clone 6, and pPS16_009 clone 4 extraction By Terrence
The extraction was carried out following the usual protocol
Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB By 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(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
5min
Hold
4°C
$\infinity\$
Primers used were:
Matrix
plasmid pJET coding sg-Nm
plasmid pJET coding FRB
Primers
pJET R and pJET F
pJET R and pJET F
Tm
60.0C
60.0C
t
15 sec
15 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)
FRB
374
sg-Nm
362
We can't see anything, the only visibles strands are the priers sequence, The result is not satisfying
Q5 high fidelity PCR of ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004 By mathilde
Fragment 1 (pPS16_001-pPS16_002) and fragment 2 (pPS16_003-pPS16_004E) were each tested three times (A,B,C) according to the following protocol :
10µL of Q5 buffer
1µL of dNTPs (10mM)
Primers mix (1µL each)
1µL of ligation product
0,25 µL of Q5 high fidelity polymerase
37,75 µL of nuclease free water The primers used were IPS83 and IPS 122 for the fragment 1, and IPS 84 and 123 for the fragment 2.
The PCR programm featured 30s of initial denaturation, 72°c of annealing temperature for 30s and 2min of final extension.
Gel electrophoresis of Q5 PCR ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004 PCR products were put to migration following the usual protocol.
PCR products
Expected band size (bp)
pPS16_001-pPS16_002
1920
pPS16_003-pPS16_004
1831
Only pPS16_001-pPS16_002 ligation product shows strands at the expected size.
Ligation of pPS16_003-pPS16_004 By Mathilde
4 µL of pPS16_003 plasmid
4 µL of pPS16_004 plasmid
1µL of T4 Buffer 10X
1µL of T4 Ligase The solution is put in incubation at 4°c over night.
