Difference between revisions of "Team:Paris Saclay/Notebook/September/6"
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= Tuesday 6<sup>th</sup> September= | = Tuesday 6<sup>th</sup> September= | ||
| − | + | ||
===Visualization=== | ===Visualization=== | ||
====Q5 PCR on dCas9 ST - GFP 11 in pSB1C3, FRB in pJET and GFP 1.9 in pUC19 ==== | ====Q5 PCR on dCas9 ST - GFP 11 in pSB1C3, FRB in pJET and GFP 1.9 in pUC19 ==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
Q5 PCR was performed on plasmids with the following protocol: | Q5 PCR was performed on plasmids with the following protocol: | ||
For each 50μl of reaction, mix the following reagents : | 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, | + | * 0,5 µL of Q5 high fidelity polymerase |
| − | * 35, | + | * 35,5 µL of nuclease free water |
| − | Mix gently and aliquot | + | Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. |
Perform PCR as follow: | Perform PCR as follow: | ||
{| class="wikitable" | {| class="wikitable" | ||
| Line 47: | Line 48: | ||
|} | |} | ||
| − | + | [[Team:Paris_Saclay/Experiments#primers|Primers]] used were: | |
{| class="wikitable" | {| class="wikitable" | ||
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====PCR Clean-up of PCR products ==== | ====PCR Clean-up of PCR products ==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]]. | PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]]. | ||
====NanoDrop Measurements==== | ====NanoDrop Measurements==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
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====Gel of cleaned up PCR products==== | ====Gel of cleaned up PCR products==== | ||
| − | ''By Maxence'' | + | ''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. | 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. | ||
| Line 120: | Line 121: | ||
|- | |- | ||
|GFP 1.9 | |GFP 1.9 | ||
| − | | | + | |862 |
|} | |} | ||
| − | [[File:T--Paris Saclay-- | + | [[File:T--Paris Saclay--Gel111.png|400px|thumb|center|Result of the migration]] |
| − | + | 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==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
PCR product GFP 11 - pSB1C3 has been linearized for further Gibson application by using DpnI treatment : | 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 [[Team:Paris_Saclay/Experiments#Purification|protocol]]. | The mix was placed 10 minutes at 37°C and was cleaned by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|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 [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. | ||
| + | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 17:02, 9 October 2016
