Line 13:
Line 13: * 2.5 µL DreamTaq Buffer
* 2.5 µL DreamTaq Buffer
* 0.5 µL of dNTPs (10mM)
* 0.5 µL of dNTPs (10mM)
− * 0.5 µL of each primer mix (10µM)
+ * 0.5 µL of each primer(10µM)
* 0.13 μl of DreamTaq Pol
* 0.13 μl of DreamTaq Pol
Line 39:
Line 39: |Final Extension
|Final Extension
|72°C
|72°C
− |7min
+ |5min
|-
|-
|Hold
|Hold
Line 73:
Line 73: !Expected band size (bp)
!Expected band size (bp)
|-
|-
− |dCas9 NM - GFP 10 - pSB1C3
+ |FRB
− |3688
+ |374
+ |sg -Nm
+ |362
|}
|}
− [[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]]
−
− GEL GEL GEL
[[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]]
[[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]]
+
+
+ [[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]]
{{Team:Paris_Saclay/notebook_footer}}
{{Team:Paris_Saclay/notebook_footer}}
Revision as of 15:31, 20 September 2016
Thuersday 25th September Lab work Visualization 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(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
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)
FRB
374
sg-Nm
362
We can't see anything, the only visibles strands are the priers sequence, The result is not satisfying
