standard pcr protocol - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki
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.
Normally, we do not prepare the PCR mix for each PCR, but we use a frozen 2xPCR 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.
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.
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.
If you have the sequence of the template and primers, PydnaTools 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 most 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).
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
may improve results:
- DMSO (up to 10%)
- Detergents (NP40, Triton X-100, Tween® 20) up to 1%
See links below for more suggestions.
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.
Mix 2.0331 g MgCl2 (MgCl2x6H2O, MW 203.31g/mol) with 50 mL of the purest available water. The final concentration is 200 mM.