Standard PCR protocol
This is a compilation of general guidelines for PCR. Most of it was lifted from the FERMENTAS website many years ago.
| Reagent stock | Final concentration |
|---|---|
| Sterile deionized water | - |
| 10X Taq buffer | 1X |
| 2-10 mM dNTP mix | 0.2 mM of each |
| Taq DNA Polymerase | 1.25 U / 50 µl |
| 25-50 mM MgCl2 | 2 mM |
| Primer I | 0.5 µM |
| Primer II | 0.5 µM |
| Template DNA | 10 pg-1 µg |
Note the concentrations of the stock solutions.
Mastermix
Normally, we do not prepare the PCR mix for each PCR, but we use a frozen 2x PCR mastermix with 2x concentration. This means that you take for example 25 µL mix to a 50 µL reaction and then add primers, template and water to 50 µL. This reduces pipetting errors and increases consistency.
Mastermix with Phusion DNA polymerase
For 1400 µL (fits in one 1.5mL Eppendorf tube!):
| Component | Volumes |
|---|---|
| H2O | 672.0 |
| 5xHF Buffer w 7.5mM MgCl2 | 560.0 |
| dNTPs(10 mM) | 56.00 |
| DMSO(100%) | 84.00 |
| Phusion (2U/µL) | 28.00 |
See this spreadsheet PhusionMasterMix2x
We add 3% DMSO for safe measure.
Mastermix with homemade Taq DNA polymerase
For 1000 µL :
| Component | Volumes |
|---|---|
| H2O | 686 |
| Taq buffer w NH4SO4 | 280 |
| MgCl2(25 mM) | 224 |
| dNTPs(10 mM) | 56 |
| DMSO(100%) | 84 |
| Taq (1U/µL) | 70 |
See this spreadsheet TaqMasterMix2x
We add 3% DMSO for safe measure.
PCR program
If you have the sequence of the template and primers, pydnaweb can simulate the PCR and suggest a PCR program.
Generally, the rate of DNA synthesis by Taq DNA Polymerase and Pfu DNA Polymerase is highest at 70-75°C. As a rule, the extension step with Taq DNA Polymerase is 1 min at 72°C for PCR products up to 2 kb.
For larger products, the extension time can be prolonged by 1 min/kb. Since Pfu DNA Polymerase exhibits lower extension rate, an extension step of 2 min/kb at 72°C is recommended.
Melting temperature (Tm):
Melting temperatures for oligonucleotides can be calculated from simple formulae (e.g. Tm = 2[A+T] + 4[G+C]), or determined by software programs developed specifically for this purpose. It is unclear which is the best method to mos accurately calculate Tm. Optimal annealing temperature (Ta):
A general rule of thumb is to use a temperature approximately 5 °C lower than the Tm of the primers. Alternatively, there are more complex equations to calculate Ta (Rychlik et al., Nucleic Acids Research 18:6409-6412).
Stock solutions:
Primers are diluted to 100µM stock if nothing else is specified. You should not use the stocks directly, make a 10 times dilution for yourself. Make sure to
MgCl2 stock solutions are typically 25mM or 50mM.
Primers: 10uM (10x dilution of the MWG standard conc) dNTPs 1 mM (10x dilution of the normal stock) [rel://protocols/Screenshot-Java-applet for PCR or qPCR reaction mixture setup - Mozilla Firefox.png]
Volume calculators: http://primerdigital.com/Tools/ReactionMixture.html Troubleshoot PCR: http://www.med.yale.edu/genetics/ward/tavi/Trblesht.html
Additives
may improve results:
- DMSO (up to 10%)
- Detergents (NP40, Triton X-100, Tween® 20) up to 1%
See links below for more suggestions.
Buffer
Fermentas (now ThermoFisher) describe a buffer that works very well in our hands. The recipe is taken from this website.
10x Taq Buffer with (NH4)2SO4 w/o MgCl2:
- 750 mM Tris-HCl (pH 8.8 at 25°C)
- 200 mM (NH4)2SO4
- 0.1% (v/v) Tween 20
This buffer can be prepared in this way (100 mL):
- Add 9.0855 g Tris-Base (MW 121.14) to about 75 mL of the purest available water and a magnetic stirrer bar.
- Set pH to 8.8 with concentrated HCl. Set the pH before the addition of the (NH4)2SO4.
- Add 2.6428 g (NH4)2SO4 (MW 132.14) to the solution and mix.
- Transfer to a 100mL volumetric flask and adjust the volume.
- Add 100µL or 90 mg Tween 20
- Mix, aliquot and freeze. Autoclaving is not needed.
This buffer has no MgCl2.
MgCl2
Mix 2.0331 g MgCl2 (MgCl2x6H2O, MW 203.31g/mol) with 50 mL of the purest available water. The final concentration is 200 mM.