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Line 125: [[File:T--Paris Saclay--090616--Gel1.jpeg|400px|thumb|center|Result of the migration]]
[[File:T--Paris Saclay--090616--Gel1.jpeg|400px|thumb|center|Result of the migration]]
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− GEL 1
No PCR products were obtained for GFP 1.9. Concerning FRB and GFP 11 - pSB1C3 amplification, good results were obtained and clones 9 for FRB and clone 8 for GFP 11 - pSB1C3 were selectionned.
No PCR products were obtained for GFP 1.9. Concerning FRB and GFP 11 - pSB1C3 amplification, good results were obtained and clones 9 for FRB and clone 8 for GFP 11 - pSB1C3 were selectionned.
Revision as of 16:34, 19 September 2016
Tuesday 6th September Lab work Visualization Q5 PCR on dCas9 ST - GFP 11 in pSB1C3, FRB in pJET and GFP 1.9 in pUC19 By Maxence & Mahnaz
Q5 PCR was performed on plasmids with the following protocol:
For each 50μl of reaction, mix the following reagents :
1 µL of plasmid
1 µL of dNTPs (10mM)
1 µL of each primer mix (10µM)
10 µL of Q5 buffer (5X)
0,5 µL of Q5 high fidelity polymerase
35,5 µL of nuclease free water Mix gently and aliquot 50 μl 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
10sec
Tm
20sec
72°C
t
Final Extension
72°C
2min
Hold
4°C
$\infty$
Primers used were:
Matrix
dCas9 ST - GFP 11 in pSB1C3 clones 6 and 8
FRB in pJET clones 4 and 9
GFP 1.9 in pUC19
Primers
iPS152 and iPS151
iPS149 and iPS150
iPS84 and iPS140
Tm
57,5°C
56,7°C
72°C
t
1 min 30
20 sec
30 sec
PCR Clean-up of PCR products By Maxence & Mahnaz
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol .
NanoDrop Measurements By Maxence & Mahnaz
Sample
Concentration (ng/µL)
PCR fragment GFP 11 clone 6
187.23
PCR fragment GFP 11 clone 8
156.85
PCR fragment FRB clone 4
75.67
PCR fragment FRB clone 9
246.41
PCR fragment GFP 1.9
22.06
Gel of cleaned up PCR products By Maxence & Mahnaz
After amplification, 3 µL of each cleaned up 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)
GFP 11- pSB1C3
2500
FRB
374
GFP 1.9
862
No PCR products were obtained for GFP 1.9. Concerning FRB and GFP 11 - pSB1C3 amplification, good results were obtained and clones 9 for FRB and clone 8 for GFP 11 - pSB1C3 were selectionned.
Linearization of PCR product GFP 11 - pSB1C3 from clone 8 By Maxence & Mahnaz
PCR product GFP 11 - pSB1C3 has been linearized for further Gibson application by using DpnI treatment :
30 µL of cleaned up PCR product GFP 11 - pSB1C3
4 µL of fast digest buffer
1 µL of DpnI
5 µL of water The mix was placed 10 minutes at 37°C and was cleaned by using the NucleoSpin Gel and PCR Clean-up kit protocol .
Transformation of DH5a cells with dCas9 NM - GFP 10 in PSB1C3 (pPS16_016) obtained by Gibson By Maxence & Mahnaz
Dh5a cells were transformed with pSB1C3 containing dCas9 NM - GFP 10 (pPS16_016) (obtained by Gibson of fragment 1 PCR products (one with Q5 Pol and one with Phusion) + fragment 2 PCR products (one with Q5 Pol and one with Phusion) + pSB1C3), or containing control (no mix added when Gibson performed) using the usual protocol .
