Difference between revisions of "Team:Paris Saclay/Notebook/September/6"
(→Q5 PCR on dCas9 ST - GFP 11 in pSB1C3, FRB in pJET and GFP 1.9 in pUC19) |
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[[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]] | [[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]] | ||
| − | GEL | + | 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. | ||
====Linearization of PCR product GFP 11 - pSB1C3 from clone 8==== | ====Linearization of PCR product GFP 11 - pSB1C3 from clone 8==== | ||
Revision as of 17:15, 13 September 2016
Contents
- 1 Tuesday 6th September
- 1.1 Lab work
- 1.1.1 Visualization
- 1.1.1.1 Q5 PCR on dCas9 ST - GFP 11 in pSB1C3, FRB in pJET and GFP 1.9 in pUC19
- 1.1.1.2 PCR Clean-up of PCR products
- 1.1.1.3 NanoDrop Measurements
- 1.1.1.4 Gel of cleaned up PCR products
- 1.1.1.5 Linearization of PCR product GFP 11 - pSB1C3 from clone 8
- 1.1.1.6 Transformation of DH5a cells with dCas9 NM - GFP 10 in PSB1C3 obtained by Gibson
- 1.1.1 Visualization
- 1.1 Lab work
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
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
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
NanoDrop Measurements
By Maxence
| 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
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 |
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.
Linearization of PCR product GFP 11 - pSB1C3 from clone 8
By Maxence
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 obtained by Gibson
By Maxence
Dh5a cells were transformed with pSB1C3 containing dCas9 NM - GFP 10 (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.
