Difference between revisions of "Team:Paris Saclay/Notebook/September/7"
(→Wednesday 7th September) |
(→Lab work) |
||
| (14 intermediate revisions by 3 users not shown) | |||
| Line 1: | Line 1: | ||
| + | {{Team:Paris_Saclay/notebook_header}} | ||
| + | |||
= Wednesday 7<sup>th</sup> September= | = Wednesday 7<sup>th</sup> September= | ||
| − | + | ||
===Visualization=== | ===Visualization=== | ||
====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 ==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
| − | Gibson was performed with cleaned up PCR products FRB from clone 9 (insert) x pSB1C3 - GFP 11 from clone 8 (plasmid) (obtained the 6th | + | 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: |
For each 20μl of reaction, mix the following reagents : | For each 20μl of reaction, mix the following reagents : | ||
| Line 16: | Line 18: | ||
Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). The PCR was performed as follow : 1 hour at 50°C. | Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). 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 (pPS16_019) obtained by Gibson==== |
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
| − | Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11, or | + | Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11 (pPS16_019), or controls (no buffer mix and plasmid alone) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. |
| − | ====Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3==== | + | ====Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3 (pPS16_016)==== |
| − | ''Maxence'' | + | ''By Maxence & Mahnaz'' |
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 [[Team:Paris_Saclay/Experiments##Polymerase_chain_reaction|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. | 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 [[Team:Paris_Saclay/Experiments##Polymerase_chain_reaction|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. | ||
| Line 47: | Line 49: | ||
|30 sec | |30 sec | ||
|- | |- | ||
| − | | | + | |63.6°C |
|30 sec | |30 sec | ||
|- | |- | ||
|72°C | |72°C | ||
| − | | | + | |1 min 30 sec |
|- | |- | ||
|Final Extension | |Final Extension | ||
| Line 62: | Line 64: | ||
|} | |} | ||
| − | + | [[Team:Paris_Saclay/Experiments#primers|Primers]] used were: | |
{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
!Matrix | !Matrix | ||
| − | !Clones containing dCas9 NM - GFP 10 in pSB1C3 | + | !Clones containing dCas9 NM - GFP 10 in pSB1C3 (pPS16_016) |
|- | |- | ||
|Primers | |Primers | ||
|iPS83 and iPS84 | |iPS83 and iPS84 | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
|} | |} | ||
| Line 92: | Line 88: | ||
|} | |} | ||
| − | [[File:T--Paris Saclay-- | + | [[File:T--Paris Saclay--Gel112.png|400px|thumb|center|Result of the migration - Phusion]] |
| + | [[File:T--Paris Saclay--Gel113.png|400px|thumb|center|Result of the migration - Q5 Pol]] | ||
| − | + | Only PCR products around 650 pb were obtained for both Pol. This kind of results has already been obtained by the past and the experience has to be improved. | |
====Samples preparation for sequencing==== | ====Samples preparation for sequencing==== | ||
| − | '' | + | ''By Maxence & Mahnaz'' |
| + | |||
| + | 20 µL of plasmides dCas9 ST - GFP 11 (pPS16_016) (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. | ||
| − | + | {{Team:Paris_Saclay/notebook_footer}} | |
Latest revision as of 17:03, 9 October 2016
