Difference between revisions of "Team:Paris Saclay/Notebook/August/16"
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= Tuesday 16<sup>th</sup> August= | = Tuesday 16<sup>th</sup> August= | ||
==Lab work== | ==Lab work== | ||
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''By Charlène'' | ''By Charlène'' | ||
| − | The PCR was carried out with a new protocol : | + | The PCR was carried out with a new protocol: |
| + | '''Q5 PCR recipe''' | ||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | |Buffer Q5 HF (5X) | ||
| + | |10µL | ||
| + | |- | ||
| + | |dNTP (10mM) | ||
| + | |1µL | ||
| + | |- | ||
| + | |Primers (each) | ||
| + | |2,5µL | ||
| + | |- | ||
| + | |DNA | ||
| + | |1µL for plasmid, 2µL for ligation's products | ||
| + | |- | ||
| + | |Q5 DNA polymerase | ||
| + | |0.25µL | ||
| + | |- | ||
| + | |Nuclease-free water | ||
| + | |up to 50µL | ||
| + | |} | ||
| − | The specific [[Team:Paris_Saclay/Experiments#primers|primers]] for each parts were using. The products were put to migrated on a 0.8%agarose gel with BET. | + | 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 | ||
| + | |$\infty$ | ||
| + | |} | ||
| + | |||
| + | The specific [[Team:Paris_Saclay/Experiments#primers|primers]] for each parts were using. | ||
| + | |||
| + | The products were put to migrated on a 0.8%agarose gel with BET. | ||
Revision as of 10:13, 16 August 2016
Contents
Tuesday 16th August
Lab work
Visualization
2.1-2.2 and 3.1-3.2 ligation
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 purify PCR products, 8µL of 3.2 purify 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
| Buffer Q5 HF (5X) | 10µL |
| dNTP (10mM) | 1µL |
| Primers (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 :
| Step | Temperature | Time |
|---|---|---|
| Initial denaturation | 98°C | 30sec |
| 30 cycles | 98°C | 5sec |
| Tannealing | 30sec | |
| 72°C | 30sec/kb | |
| Final Extension | 72°C | 2min |
| Hold | 4°C | $\infty$ |
The specific primers for each parts were using.
The products were put to migrated on a 0.8%agarose gel with BET.
