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Team:Cambridge/PCRprotocol
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CharlotteBG (Talk | contribs) (→PCR using Phusion DNA polymerase:) |
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==PCR using Phusion DNA polymerase:== | ==PCR using Phusion DNA polymerase:== | ||
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<big>If this reaction is to be done from scratch, add the reagents in the order they are listed in the table. When multiple PCR experiments need to be run concurrently, everything that is not experiment specific (primers and template DNA) can be made up ahead of time as a master mix. In this case the master mix should be kept on ice as it contains enzymes. The primers and template DNA should be added to a PCR tube and the master mix added just before the sample is put in just before PCR begins. </big> | <big>If this reaction is to be done from scratch, add the reagents in the order they are listed in the table. When multiple PCR experiments need to be run concurrently, everything that is not experiment specific (primers and template DNA) can be made up ahead of time as a master mix. In this case the master mix should be kept on ice as it contains enzymes. The primers and template DNA should be added to a PCR tube and the master mix added just before the sample is put in just before PCR begins. </big> | ||
Revision as of 15:30, 12 July 2012
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PCR using Phusion DNA polymerase:
If this reaction is to be done from scratch, add the reagents in the order they are listed in the table. When multiple PCR experiments need to be run concurrently, everything that is not experiment specific (primers and template DNA) can be made up ahead of time as a master mix. In this case the master mix should be kept on ice as it contains enzymes. The primers and template DNA should be added to a PCR tube and the master mix added just before the sample is put in just before PCR begins.
| Reagent | 50 µl reaction | 20 µl reaction | Final Concentration |
| H2O | Add to make 50 µl | Add to make 20 µl | |
| 5x Phusion buffer | 10 µl | 4 µl | 1x |
| 10 mM dNTPs | 1 µl | 0.4 µl | 200 µM |
| Primer 1 | x µl | x µl | 0.5 µM |
| Primer 2 | x µl | x µl | 0.5 µM |
| Template DNA | x µl | x µl | |
| Phusion DNA polymerase | 0.5 µl | 0.2 µl | 0.02 u/ µl |
N.B. The volume of primer that needs to be added will vary in accordance with the concentration of DNA in the initial sample.
PCR machine settings:
| Temperature (oc) | Time (s) | ||
| Hold 1 | 95 | 360 | |
| Cycle (30x) | Denaturing | 98 | 10 |
| Annealing | 60 | 30 | |
| Elongation | 72 | 120 | |
Safety considerations:No bacteria are used during the reaction there is therefore little or no biological hazard. Nevertheless, it is important to observe correct laboratory procedure and wear appropriate clothing and gloves. PCR occurs at high temperature, and this may present a risk, depending on the PCR machine employed.
