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	1 µM
Primer II	1 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 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.

See here and here

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):

1. Add 9.0855 g Tris-Base (MW 121.14) to about 75 mL of the purest available water and a magnetic stirrer bar.
2. Set pH to 8.8 with concentrated HCl. Set the pH before the addition of the (NH4)2SO4.
3. Add 2.6428 g (NH4)2SO4 (MW 132.14) to the solution and mix.
4. Transfer to a 100mL volumetric flask and adjust the volume.
5. Add 100µL or 90 mg Tween 20
6. 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.