Line 76:
Line 76: GEL GEL GEL
GEL GEL GEL
+ Séquençage
− all 4 plasmids were sent for sequencing.
+ ====Samples preparation for sequencing====
+ ''by Mahnaz''
+
+ 15 µL of plasmides plasmids coding FRB from colonies 4,5,9and 12 were sent to be sequenced. 20 µL of the primers pJET F (5µM) and pJET R (5µM) were sent for sequencing.
====NanoDrop Measurements====
====NanoDrop Measurements====
Revision as of 16:33, 20 September 2016
Monday 29th August Lab work Visualization By Mahnaz
2.5 µL DreamTaq Buffer
0.5 µL of dNTPs (10mM)
1 µL of each primer (10µM) primer pJET R and F
0.13 μl of DreamTaq Pol
20 µl H2O PCR was performed as follow:
Step
Temperature
Time
Initial denaturation
95°C
3 min
30 cycles
95°C
30 sec
52
30 sec
72°C
1 min
Final Extension
72°C
7min
Hold
4°C
\infinity\
Primers used were:
Matrix
Clones containing dCas9 NM - GFP 10 in pSB1C3
Primers
pJET R and F
Tm
60°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)
FRB in pJET
474
GEL GEL GEL
Séquençage
Samples preparation for sequencing by Mahnaz
15 µL of plasmides plasmids coding FRB from colonies 4,5,9and 12 were sent to be sequenced. 20 µL of the primers pJET F (5µM) and pJET R (5µM) were sent for sequencing.
NanoDrop Measurements By Mahnaz
Sample
Concentration (ng/µL)
PCR fragment FRB clone 4
58.43
PCR fragment FRB clone 5
320.75
PCR fragment FRB clone 9
830.99
PCR fragment FRB clone 12
473.37
