Difference between revisions of "Team:Paris Saclay/Notebook/September/9"
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= Friday 9<sup>th</sup> September= | = Friday 9<sup>th</sup> September= | ||
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===Visualization=== | ===Visualization=== | ||
| − | ====Colony PCR of 16 clones containing FRB - GFP 11 in pSB1C3==== | + | ====Colony PCR of 16 clones containing FRB - GFP 11 in pSB1C3 (pPS16_019)==== |
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
Colonies were obtained for the Gibson done the 7th September but not for the one done the 8th September. A colony PCR was done for 16 clones from the 7th September. For that purpose, 16 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. | Colonies were obtained for the Gibson done the 7th September but not for the one done the 8th September. A colony PCR was done for 16 clones from the 7th September. For that purpose, 16 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. | ||
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| − | + | [[Team:Paris_Saclay/Experiments#primers|Primers]] used were: | |
{| class="wikitable" | {| class="wikitable" | ||
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!Matrix | !Matrix | ||
| − | !Clones containing FRB - GFP 11 in pSB1C3 | + | !Clones containing FRB - GFP 11 in pSB1C3 (pPS16_019) |
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|Primers | |Primers | ||
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| − | + | [[File:T--Paris Saclay--Gel117.png|400px|thumb|center|Result of the migration]] | |
| − | All PCR products were at the good size and seemed to be concentrated. It could be explained as these clones were grown for 2 days. Clones | + | All PCR products were at the good size and seemed to be concentrated. It could be explained as these clones were grown for 2 days. Clones 4, 9, 13 and 16 were selected and were grown at 37°C overnight. |
====PCR on GFP 1.9 in pUC19, gblock 1.1, gblock 1.2, gblock 2.1 and gblock 2.2 with 3% DMSO ==== | ====PCR on GFP 1.9 in pUC19, gblock 1.1, gblock 1.2, gblock 2.1 and gblock 2.2 with 3% DMSO ==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
As the annealing temperature (Tm) seems too high to obtain good results for these amplifications, DMSO was used in order to reduce the annealing temperature during the PCR. For that purpose, PCR was performed on plasmids with the following protocol.For each amplification, two matrix were used: one was a previous PCR products and one was a plasmid. | As the annealing temperature (Tm) seems too high to obtain good results for these amplifications, DMSO was used in order to reduce the annealing temperature during the PCR. For that purpose, PCR was performed on plasmids with the following protocol.For each amplification, two matrix were used: one was a previous PCR products and one was a plasmid. | ||
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| − | + | [[Team:Paris_Saclay/Experiments#primers|Primers]] used were: | |
{| class="wikitable" | {| class="wikitable" | ||
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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. | ||
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| − | + | [[File:T--Paris Saclay--Gel118.png|400px|thumb|center|Result of the migration]] | |
All PCR products were at the good size. | All PCR products were at the good size. | ||
====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]]. For each amplification, only PCR products from plasmids were cleaned in order to reduce the risks of mutations. For GFP 1.9 amplification products, the 4 samples were pooled before clean up. | + | PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]]. For each amplification, only PCR products from plasmids were cleaned in order to reduce the risks of mutations. For GFP 1.9 amplification products, the 4 samples were pooled before clean up. |
====Results about FRB - GFP 11 transformed colonies from 8th September==== | ====Results about FRB - GFP 11 transformed colonies from 8th September==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
During the afternoon, observations were made about the 8th September Gibson, but as results were obtained for the 7th September Gibson, a colony PCR was not run. Nevertheless, different conditions were tested, and a counting of the colonies was done: | During the afternoon, observations were made about the 8th September Gibson, but as results were obtained for the 7th September Gibson, a colony PCR was not run. Nevertheless, different conditions were tested, and a counting of the colonies was done: | ||
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| + | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 17:04, 9 October 2016
