Difference between revisions of "Team:Paris Saclay/Notebook/August/16"
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{{Team:Paris_Saclay/notebook_header}} | {{Team:Paris_Saclay/notebook_header}} | ||
= Tuesday 16<sup>th</sup> August= | = Tuesday 16<sup>th</sup> August= | ||
| − | + | ||
===Visualization=== | ===Visualization=== | ||
| − | ==== | + | ==== 2.1-2.2 and 3.1-3.2 ligation ==== |
''By Charlène'' | ''By Charlène'' | ||
| − | + | 8µL of 2.1 purify PCR products, 8µL of 2.2 purify PCR products, 2µL of ligase T4 Buffer and 2L of ligase T4 were mixed. 8µL of 3.1 purified PCR products, 8µL of 3.2 purified PCR products, 2µL of ligase T4 Buffer and 2µL of ligase T4 were mixed. They were incubated for 1h at RT. | |
| + | |||
| + | ==== Q5 PCR on the ligation products and pPS16_008 clones 1 and 2==== | ||
| + | ''By Charlène'' | ||
| + | |||
| + | The PCR was carried out with a new protocol: | ||
| + | '''Q5 PCR recipe''' | ||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | |Buffer Q5 HF (5X) | ||
| + | |10µL | ||
| + | |- | ||
| + | |dNTP (10mM) | ||
| + | |1µL | ||
| + | |- | ||
| + | |Primers (10 µM, each) | ||
| + | |2,5µL | ||
| + | |- | ||
| + | |DNA | ||
| + | |1µL for plasmid, 2µL for ligation's products | ||
| + | |- | ||
| + | |Q5 DNA polymerase | ||
| + | |0.25µL | ||
| + | |- | ||
| + | |Nuclease-free water | ||
| + | |up to 50µL | ||
| + | |} | ||
| + | |||
| + | Steps for PCR : | ||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | !Step | ||
| + | !Temperature | ||
| + | !Time | ||
| + | |- | ||
| + | |Initial denaturation | ||
| + | |98°C | ||
| + | |30sec | ||
| + | |- | ||
| + | |rowspan="3"|30 cycles | ||
| + | |98°C | ||
| + | |5sec | ||
| + | |- | ||
| + | |T<sub>annealing</sub> | ||
| + | |30sec | ||
| + | |- | ||
| + | |72°C | ||
| + | |30sec/kb | ||
| + | |- | ||
| + | |Final Extension | ||
| + | |72°C | ||
| + | |2min | ||
| + | |- | ||
| + | |Hold | ||
| + | |4°C | ||
| + | |$\infty$ | ||
| + | |} | ||
| + | |||
| + | An specific [[Team:Paris_Saclay/Experiments#primers|primers]] for each part was used. | ||
| + | |||
| + | The products were migrated on a 0.8% agarose gel with BET. | ||
| + | [[File:T--Paris Saclay--20160816 fragments 2, 3 and gblock 4.1.JPG|500px|thumb|right|Gel Electrophoresis of PCR products fragments 2, 3 and gblock 4.1.JPG]] | ||
| + | All the bands have the good size so the PCR products were purify | ||
| + | ==== PCR Clean-up with the NucleoSpin kit ==== | ||
| + | ''By Mahnaz'' | ||
| + | _ | ||
| + | The purification was carried out on PCR products pSB1C3 (linearized) and 4.2 also the ligation products pPS16_003-pPS16_004 and pPS16_005-pPS16_006 following the usual [[Team:Paris_Saclay/Experiments#Purification|protocol]]. | ||
| + | [[File:T--Paris_Saclay--180816_visualization_PCR_CleanUp.jpg|400px|thumb|right|Result of the PCR]] | ||
| + | ==== 4.1-4.2 ligation ==== | ||
| + | ''By Mahnaz'' | ||
| + | 8 µL of 4.1 purified PCR product, 8µL of 4.2 purified PCR product, 2 µL of ligase T4 Buffer and 2 µL of ligase T4 were mixed. The ligation reaction mixture were incubated overnight at 4°C. | ||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 16:42, 9 October 2016
