A basic PCR protocol consists of cycles of three steps:
The exact temperature and timing of each step may vary depending on the polymerase being used (such as Taq or Phusion). PCR protocols can also vary depending on the template: purified plasmid or genomic DNA is typical but PCRs can also be performed on DNA released directly from bacterial liquid cultures or colonies from plates.
Tip: Use a Mastermix: When more than a few reactions are being made (5+), it is advisable to use a mastermix. A mastermix is a premixed batch of all of the ingredients common to all of the reactions (e.g., water, buffer, polymerase, dNTPs). To make a mastermix, take the amount of each of the individual ingredients needed (ex: 3 μl 10× dNTPs) and multiply it by (number of reactions + 1). Remember to include controls in the total reaction count. So if 8 total reactions are being run, the mastermix would contain 27 μl 10× dNTPs. When many PCR reactions are being run (15+) it often becomes necessary to add more than just 1 extra batch for the final volume. In this case, you can add an extra 10% of each component. Mastermixes not only save time, but they also allow for much greater precision in mixing the right amounts of PCR components. Measuring out 0.3 μl of 100× Taq polymerase with a pipette is very inaccurate, however, 2.7 μl of the same Taq can go into the mastermix, a volume that can be pipetted with much higher precision. When making a mastermix be sure to add enzyme last so that it only experiences proper buffer conditions.
A typical reaction has a final volume of 30 μl, a template concentration of 0.1ng/μl, and primer concentrations of 500nM each. This chart shows the volumes of various ingredients that should be used. These PCR conditions are suitable for products ranging up to 3 kb in length. PCRs that result in longer products may require optimization of the dNTP and primer concentrations and the use of special DNA polymerase kits.
Template | 10µM UF primer | 10µM DR primer | 10x buffer | 2mM dNTPs | 5U/µL taq | ddH2O | Total |
---|---|---|---|---|---|---|---|
X | 1.5 | 1.5 | 3 | 3 | 0.3 | 20.7 - X | 30 μl |
10x dNTPs are 2 mM in each dNTP.
If the stock concentration of DNA is 1ng/μl, 3 μl would give the desired concentration of template. 1.5 μl of a 10μM primer gives a final concentration of 500nM. Water is added as needed to create a final volume of 30 μl.
In the thermocycler, make sure that the program "PCR" contains the appropriate temperatures and times. The most important step to check, is that the 72°C elongation step should be roughly 1 minute for every 1000bp of the longest PCR product. If the longest expected product is 2500bp, then 2.5 minutes at 72°C should be appropriate. For most PCRs, 30-40 cycles should be appropriate.
Further Reading
When using Phusion polymerase, use the NEB calculator to approximate your annealing temperatures.
For a 20 ul reaction
Template | 10µM UF primer | 10µM DR primer | 5x Phusion buffer | 10mM dNTPs | Phusion poly | ddH2O | Total |
---|---|---|---|---|---|---|---|
X | 1.0 | 1.0 | 4 | 0.4 | 0.2 | up to 20ul | 20 μl |
For a 50 ul reaction
Template | 10µM UF primer | 10µM DR primer | 5x Phusion buffer | 10mM dNTPs | Phusion poly | ddH2O | Total |
---|---|---|---|---|---|---|---|
X | 2.5 | 2.5 | 10 | 1 | 0.5 | up to 50µl | 50 μl |
Final Concentrations
Template | For primer | Rev primer | 5x Phusion buffer | 10mM dNTPs | Phusion poly | ddH2O |
---|---|---|---|---|---|---|
<250 ng | 0.5µM | 0.5µM | 1x | 200µM | 1.0 units/50ul PCR | - |
NEB recommends a template amount (for 50µl reaction) of 50 ng - 250 ng for genomic DNA and 1 pg - 10 ng for plasmid or viral DNA.
Although the recommended Phusion polymerase concentration is 1.0 units/50 ul rxn (20 units/ml) this concentration may have to be changed based on amplicon length and difficulty. The concentration may vary from (0.5-2 units/50 ul rxn) but should not exceed 2 units/50 ul rxn, especially for amplicons longer than 5 kb.
Thermal cycler conditions:
Notes:
In order to analyze PCR results, the products are run on an agarose gel and the resulting gel is observed in UV light.
First, the gel has to be made. A standard 1% agarose gel uses 1g of agarose for every 100 ml of buffer. A different percentage may be used, and gels with less than 1% agarose may be used to clearly distinguish products of very similar sizes. For a standard 50 ml gel, add .5g agarose and 50 ml TAE (1X) buffer to a 125ml flask and heat for 1:30 minutes. Meanwhile, assemble a gel rig and find a comb with an appropriate number of wells, then place the comb into the rig. After heating add 2.5 μl SYBR Safe (5μl SYBR Safe for every 100mL gel) and swirl to mix. Pour the liquid from the flask into the rig and wait about 30 minutes for it to solidify.
Once the gel has solidified, gather all PCR products that are to be run, an appropriately sized ladder, and 6x loading dye. The latter two may be found in the 4°C fridge in the computer room adjacent to the gel area. First, load 6-7 μl of ladder into the first well. The easiest way to combine dye and DNA is to cut out a 4x4 sheet of parafilm, make a drop of 1 μl of dye onto the parafilm for each sample to be run. Next, add 5 μl of PCR product to the dye and pipette up and down to homogenize. Once all samples are combined with dye, load them into the gel, making note of what sample goes into what lane.
Barrick Lab > ProtocolsStandardPCR