Difference between revisions of "Team:Paris Saclay/Notebook/September/8"
(→Gibson of cleaned up PCR products FRB from clone 9 x pSB1C3 - GFP 11 from clone 8) |
(→Gibson of cleaned up PCR products FRB from clone 9 x pSB1C3 - GFP 11 from clone 8) |
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* 10 µL of buffer mix | * 10 µL of buffer mix | ||
| − | Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). Two different buffer mix | + | Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). Two different buffer mix were tested (the usual one and another one from Philippe). The PCR was performed as follow : 1 hour at 50°C. |
====Transformation of DH5a cells with FRB - GFP 11 in pSB1C3 obtained by Gibson==== | ====Transformation of DH5a cells with FRB - GFP 11 in pSB1C3 obtained by Gibson==== | ||
Revision as of 15:23, 13 September 2016
Thursday 8th September
Lab work
Visualization
Gibson of cleaned up PCR products FRB from clone 9 x pSB1C3 - GFP 11 from clone 8
By Maxence
As no colonies were obtained after one night culture, a new Gibson was performed with cleaned up PCR products FRB from clone 9 (insert) x pSB1C3 - GFP 11 from clone 8 (plasmid) (obtained the 6th september) with the following protocol.
Concentration of insert and plasmid were determinated again by Nano drop. For each 20μl of reaction, mix the following reagents :
- 0.65 µL of insert
- 1.1 µL of plasmid
- 8,25 µL of water
- 10 µL of buffer mix
Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). Two different buffer mix were tested (the usual one and another one from Philippe). The PCR was performed as follow : 1 hour at 50°C.
Transformation of DH5a cells with FRB - GFP 11 in pSB1C3 obtained by Gibson
By Maxence
Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11, or containing control (no mix added when Gibson performed) using the usual protocol.
Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3
Maxence
Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 16 clones containing dCas9 NM - GFP 10 obtained by Q5 amplification and 16 clones containing dCas9 NM - GFP 10 obtained by Phusion amplification were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) 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)
- 1 µL of each primer mix (10µM)
- 0.13 μl of DreamTaq Pol
PCR was performed as follow:
| Step | Temperature | Time |
|---|---|---|
| Initial denaturation | 95°C | 3 min |
| 30 cycles | 95°C | 30 sec |
| Tm | 30 sec | |
| 72°C | t | |
| Final Extension | 72°C | 7 min |
| Hold | 4°C | $\infinity\$ |
Primers used were:
| Matrix | Clones containing dCas9 NM - GFP 10 in pSB1C3 |
|---|---|
| Primers | iPS83 and iPS84 |
| Tm | 63.6°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) |
|---|---|
| dCas9 NM - GFP 10 - pSB1C3 | 3688 |
GEL GEL GEL
Samples preparation for sequencing
by Maxence
20 µL of plasmides dCas9 ST - GFP 11 (clones 6 and 8) were sent to be sequenced. 20 µL of the primers iPS168 (5µM), iPS169 (5µM) and iPS171 (5µM) were sent for sequencing.
