Difference between revisions of "PCR"
From Genetics Wiki
| Line 2: | Line 2: | ||
:H<sub>2</sub>O 10.5 µl | :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 | ||
| Line 12: | Line 12: | ||
: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 m extension (this can be raised to 5 m for several kbp long segments, some polymerases work better at a different temperature, 68 C - 72 C) | ::70 C 1 m extension (this can be raised to 5 m 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. | :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. | ||
Revision as of 15:47, 16 July 2018
For a 25 µl total volume reaction using a micropipette add to a PCR tube:
- 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 m extension (this can be raised to 5 m 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.
