Difference between revisions of "PCR"
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| − | For a 25 µl total volume reaction using a micropipette add to a PCR tube: | + | For a 25 [[µl]] total volume reaction using a micropipette add to a PCR tube: |
| − | :H<sub>2</sub>O 10.5 µl | + | :Nuclease-free H<sub>2</sub>O 10.5 [[µl]] |
| − | :Taq 2X Master Mix 12.5 µl | + | :Taq [[PCR 2X Master Mix|2X Master Mix]] 12.5 [[µl]] |
| − | :Forward primer 0.5 µl 10 µM solution | + | :Forward primer 0.5 [[µl]] 10 [[µM]] solution |
| − | :Reverse primer 0.5 µl 10 µM solution | + | :Reverse primer 0.5 [[µl]] 10 [[µM]] solution |
| − | :DNA template 1 µl (adding sample DNA last minimizes potential contamination of the other solutions) | + | :DNA template 1 [[µl]] (adding sample DNA last minimizes potential contamination of the other solutions) |
This is then placed in a thermocycler. General PCR cycling conditions are: | This is then placed in a thermocycler. General PCR cycling conditions are: | ||
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:Then 30 cycles of: | :Then 30 cycles of: | ||
::95 C 20 s melting | ::95 C 20 s melting | ||
| − | ::annealing temperature (this varies with the primers used, usually between 55 C and 65 C although some primers can be lower) 40s | + | ::annealing temperature (this varies with the primers used, it is very important to optimize this for your primers, usually between 55 C and 65 C although some primers can be lower) 40s |
| − | ::70 C 1 | + | ::70 C 1 min extension (this can be raised to 5 min for several kbp long segments, some polymerases work better at a different temperature, 68 C - 72 C) |
| − | : | + | :And a final extension of 70 C for 5 minutes. |
| − | There is a lot of optimization of these conditions based on the template DNA, primers, and polymerase enzyme used. Most commonly the annealing temperature is adjusted. Occasionally the magnesium concentration in the master mix and the extension times are also adjusted. In general there is no reason to go above 30 cycles. At this point there will theoretically be one billion copies (≈ 2<sup>30</sup>) of a single starting DNA template molecule. | + | There is a lot of optimization of these conditions based on the template DNA, primers, and polymerase enzyme used. Most commonly the annealing temperature is adjusted. Occasionally the magnesium concentration in the master mix and the extension times are also adjusted. In general there is no reason to go above 30 cycles. At this point there will theoretically be one billion copies (≈ 2<sup>30</sup>) of a single starting DNA template molecule if the reaction proceeds at maximum efficiency. |
| + | |||
| + | =Notes= | ||
| + | |||
| + | What about combining robotics and molecular genetics in high school biology to build a thermocycler (transfer tubes to water baths at different temperatures and monitor for the correct temperature)? | ||
| + | |||
| + | =What Links Here= | ||
| + | {{Special:WhatLinksHere/{{FULLPAGENAME}}}} | ||
Latest revision as of 17:35, 4 September 2018
For a 25 µl total volume reaction using a micropipette add to a PCR tube:
- Nuclease-free H2O 10.5 µl
- Taq 2X Master Mix 12.5 µl
- Forward primer 0.5 µl 10 µM solution
- Reverse primer 0.5 µl 10 µM solution
- DNA template 1 µl (adding sample DNA last minimizes potential contamination of the other solutions)
This is then placed in a thermocycler. General PCR cycling conditions are:
- An initial 95 C 30 s
- Then 30 cycles of:
- 95 C 20 s melting
- annealing temperature (this varies with the primers used, it is very important to optimize this for your primers, usually between 55 C and 65 C although some primers can be lower) 40s
- 70 C 1 min extension (this can be raised to 5 min for several kbp long segments, some polymerases work better at a different temperature, 68 C - 72 C)
- And a final extension of 70 C for 5 minutes.
There is a lot of optimization of these conditions based on the template DNA, primers, and polymerase enzyme used. Most commonly the annealing temperature is adjusted. Occasionally the magnesium concentration in the master mix and the extension times are also adjusted. In general there is no reason to go above 30 cycles. At this point there will theoretically be one billion copies (≈ 230) of a single starting DNA template molecule if the reaction proceeds at maximum efficiency.
Notes
What about combining robotics and molecular genetics in high school biology to build a thermocycler (transfer tubes to water baths at different temperatures and monitor for the correct temperature)?
What Links Here
- Methods (← links)
- ExoSAP Reaction (← links)
