Endpoint PCR Technical Guide

I. Select an optimal product to meet your PCR needs. Follow the Product Selection Guides to choose the right product:

• Cloning related applications    Endpoint PCR Technique Selection Guide
• Standard PCR    Application overview and Product Selection Guide
  
• Hot Start PCR    Application overview and Product Selection Guide
• High Fidelity, Long and GC Rich PCR   Application overview and Product Selection Guide
• Reverse Transcription and RT PCR    Application overview and Product Selection Guide

II. Prevention of PCR contamination

• Keep a clean work environment. When preparing PCR, care should be taken to eliminate the possibility of contamination with foreign DNA templates.
• Use separate clean areas for sample preparation, reaction mixture preparation and cycling.
• Before PCR setup, clean your working space with 70% ethanol or with a special DNA-removing spray.
• Always wear fresh gloves.
• Use sterile tubes and pipette tips with aerosol filters for PCR setup. Most contaminating templates come from dirty pipettes.
• Use DNA- and nuclease-free reagents, including water.
• With every PCR setup, perform a contamination-control reaction without template DNA.
• Use the advantages of Uracil-DNA Glycosylase: Amplification of contaminating templates remaining in the work environment from previous experiments is one of the most common contamination problems. The use of Uracil-DNA Glycosylase and dUTP (dNTP/dUTP Mix) helps prevent carry-over contamination. The typical workflow follows:

Uracil-DNA Glycosylase degrades DNA containing uracil. To prevent carry-over contamination, use dUTP or dTTP/dUTP rather than dTTP alone for PCR. Taq DNA polymerases incorporate dUTP efficiently. However, with proofreading polymerases, such as Pfu DNA Polymerase or Long and High Fidelity PCR Master Mix, the use of dUTP will result in lower PCR yield. dUTP does not change the electrophoretic mobility or ethidium-bromide-staining efficiency of DNA. After PCR with dUTP, the PCR products are substrates for UDG and any DNA containing uracil is degraded during the brief incubation with Uracil-DNA Glycosylase. This step eliminates contaminating DNA templates from previous experiments.

 III. Correct design and use of primers

• If possible, use appropriate software for primer design. Software will help you to avoid primer self-complementarities, multiple template–primer complementarities, and direct repeats in the primer to prevent hairpin and primer–dimer formation. Complementarity at the 3' ends causes the formation of primer–dimers and is one of the most common PCR problems.
• Design primers 20–30 nucleotides in length.
• Ideally, use primers with about 50% GC content. If possible, choose primers with one or two Gs or Cs at the 3’ end.
• For primers up to 25 bases long, calculate the melting temperature as follows: T_m = 4 (G + C) + 2 (A + T), where G, C, A, T indicate the number of each nucleotide in the primer. For longer primers, use software to calculate the melting temperature correctly.
• The melting temperature for primers used in the same reaction should be within 5°C of each other. Calculated primer melting temperature should be above 45°C.
• The purity and chemical integrity of the primers is crucial for good PCR results. Primers should always be present in excess in the reaction mixture and at equal concentrations, typically within the range of 0.1 μM to 1 μM. Optimal primer concentration is determined empirically; excessive primer concentration can reduce PCR specificity due to increased miss-priming.

IV. Template issues

• There are products available that allow PCR of samples with carry over contamination. Such reactions typically require either special robust enzyme formulations, modified enzymes, such as YourTaq Hot Start DNA Polymerase, additives or a combination of these. For standard PCR without special additives using common enzymes, such as Taq DNA Polymerase, the use of high-quality, purified DNA templates greatly enhances the success of PCR.
• Different DNA purification methods and commercial products are used for template preparation before PCR. However, some sample preparations might contain trace amounts of PCR inhibitors, such as phenol, EDTA or proteinase K. It is always helpful to remove the contaminants with ethanol precipitation and wash the DNA pellet with 70% ethanol.
• Too much template increases the risk of nonspecific PCR. Insufficient template reduces the accuracy. Optimal amounts of template DNA for a 50 µl reaction volume:

Plasmid or phage: 0.01–1 ng
Genomic: 0.1–1 µg

• DNA is the template for PCR, whereas RNA is the template for reverse transcription and RT-PCR. The first step in RT-PCR is the synthesis of a single-stranded DNA copy from the RNA template. This process employs reverse transcriptases, which are RNA-dependent DNA Polymerases, such as MMuLV Reverse Transcriptase. The synthesized single-stranded cDNA is then used as a template for PCR.

 V. dNTPs issues

• For the best PCR results, use only the highest quality (99% pure) dNTP Sets and Mixes. dNTP purity is especially important for long and high fidelity PCR. Unbalanced dNTP concentrations promote higher frequencies of miss-incorporation.
• The recommended final concentration for each dNTP is 0.2–0.25 mM. Higher concentration (up to 0.4 mM) increases yields. However, as Mg²⁺ binds to the dNTPs, the Mg²⁺ concentration should to be adjusted accordingly. High dNTP and magnesium concentrations reduce PCR fidelity.
• Thaw and mix each dNTP solution before use. This is especially important for high-concentration solutions.
• Follow the guidelines below to prepare a 1 ml dNTP mixture:

• Follow the guidelines below to prepare different mixes of all four dNTPs (2 mM, 2.5 mM, 10 mM or 25 mM dNTP) and to use the mix for a final 0.2 mM concentration in the PCR:

VI. Magnesium concentration issues

• Magnesium ions stabilize primer–template complexes. Some PCR buffers for Taq DNA Polymerase contain Mg²⁺ at optimal concentration. Other buffers do not contain magnesium (Taq DNA Polymerase, recombinant) but are provided with a separate vial of magnesium chloride, allowing different magnesium concentrations to be prepared.
• Mg²⁺ binds to dNTPs, primers and DNA template. It is highly recommended that Mg²⁺ concentration (in a range of 1–4 mM) is optimized for every PCR.
• Insufficient magnesium reduces the yield of PCR product. Excessive magnesium concentration causes non-specific PCR products and reduced PCR fidelity.
• The ratio of dNTPs and magnesium in the PCR should generally be 1:2. When dNTP concentration is increased, the magnesium concentration should be increased accordingly.
• If DNA samples contain EDTA or other metal chelators, the magnesium ion concentration should be increased accordingly, as one molecule of EDTA binds one molecule of Mg²⁺.
• Many applications work at the standard concentration of 1.5 mM MgCl₂. However advanced applications using genomic DNA require higher MgCl₂ concentrations (2–4 mM). Adjust PCR magnesium concentration with the supplied MgCl₂ solution according to the table below:

 VII. Optimal PCR enzyme concentration

• Every supplier of thermophilic polymerases indicates the optimal amount of the enzyme for PCR. Typically, this is about 0.5–1.5 units of Taq DNA Polymerase in a 50 μl reaction volume.
• For low-volume reactions and for immediate use, enzyme dilutions can be made with 1x enzyme reaction buffer.
• It is advisable to determine the optimal amount of the enzyme empirically, starting with the lowest concentration.
• Insufficient enzyme can result in low yield, as the polymerase might be inhibited by template impurities and low-quality dNTPs or primers. Excessive enzyme concentration might increase background.
• For reproducible results, it is advisable avoid pipetting volumes smaller than 0.5 µl by preparing a reaction master mix containing buffer, magnesium, dNTPs and enzyme. Alternatively, use PCR Master Mixes with optimized reagent concentrations.
• PCR setup should always be performed on ice as Taq DNA Polymerase exhibits low activity during reaction setup at room temperature. This activity can result in non-specific priming events leading to generation of nonspecific amplification products during PCR. To avoid such problems and to perform room-temperature reaction setup without a risk of background, use Hot Start DNA Polymerases or hot-start master mixes.

VIII. PCR enhancers and other additives

• Despite high-quality PCR reagents and pure template, some targets can be difficult to amplify. This can be due to high GC content or complex structures in the template or to enzyme-inhibiting contaminants. In such cases, additives promoting DNA denaturation might increase PCR product yield and specificity.
• The most common additives are 5% dimethyl sulfoxide (DMSO), formamide, 1 M betaine and 1% glycerol. BSA (bovine serum albumin) at concentrations of up to 0.8 μg/μl has been shown to bind PCR inhibitors, which might be present in the reaction mixture. If required, use commercially available additives at recommended concentrations.
• When using any PCR additive, it is very important to adjust the amount of enzyme accordingly, as additives might have inhibitory effects on thermophilic polymerases.
• biotechrabbit supplies two PCR additives (these additives are intended for use with their respective enzyme and are not available separately): PCR Booster, supplied with the Hot Start DNA Polymerases, which might increase product yield and eliminate background and PCR Helper, supplied with the Long and High Fidelity PCR Enzymes, which might improve yields from GC-rich templates.

IX. PCR cycling: denaturation

• The denaturation process in PCR can be divided into two steps: initial denaturation, which is performed at the beginning of the PCR, and the denaturation during each cycle.
• Complete initial DNA denaturation is essential for good PCR yield. Typically 1–3 minutes denaturation at 95°C is sufficient. For GC-rich templates, up to 10 minutes might be needed. If a very long initial denaturation step is performed, it is advised to add the enzyme only after the denaturation is completed.
• During cycling, the denaturation  is typically performed 30 seconds to 2 minutes at 94–95°C per cycle. For GC-rich templates, this step can be prolonged to 3–4 minutes. Alternatively, as described above, DNA denaturation can be enhanced by using additives, such as glycerol, DMSO or formamide. When additives are used, the annealing temperature must to be adjusted, as the melting temperature of the primer–template complexes might decrease significantly.

X. PCR cycling: annealing

• Primer annealing temperature should be 5°C lower than the lowest primer melting temperature.
• The annealing duration is typically 30 seconds to 2 minutes.
• Low annealing temperature leads to high background. High annealing temperatures might reduce yield.
• Calculate primer melting temperatures using appropriate software.
• Optimize annealing temperature stepwise in 1–2°C increments.
• In the presence of additives (glycerol, DMSO, formamide and betaine), the annealing temperature may need to be adjusted.

XI. PCR cycling: extension

• During PCR extension step, the primer elongation reaction is typically performed for 1 minute at 72°C, the optimal temperature for Taq DNA Polymerase, for PCR products up to 2 kb.
• For long-range PCR, the extension time is prolonged by 1 min/kb and extension temperature is lower (68^oC) to minimize loss of enzyme activity.
• As Pfu is slower than Taq DNA Polymerase, an extension step of 2 minutes per kb at 72°C is used.
• The final extension step is usually longer to ensure completion of PCR products. This final step, typically 15–30 minutes at 72^oC, is especially important when the PCR product will be cloned into the T vector.

XII. Number of PCR cycles

• Typically, 25–30 cycles are enough for a good yields.
• The optimal number of cycles should be determined according to the quantity and complexity of the template and the amplicon length.
• An insufficient number of cycles may result in poor yield.
• Too many cycles can cause high background.
• When the amount of template is very low, 10 copies for example, 40 cycles is recommended.

XIII. Correct analysis and quantification of PCR products

• PCR products must be purified before spectrophotometric measurement, as the presence of the primers and dNTPs in the reaction mixture interferes with absorbance measurements.
• For approximate quantification of PCR product, standard agarose gel electrophoresis and ethidium bromide staining is most commonly used. By using appropriate DNA Ladders and DNA Loading Dyes, the size and approximate quantity of the PCR product can be determined by comparing the intensity of the band with the ladder band of known quantity that is closest in size.
• If PCR is performed with standard reagents containing no dyes, DNA Loading Dye to PCR should be added to products prior loading onto a gel.
• To save time and effort, special enzymes, buffer and master mix formulations that contain electrophoresis loading dyes can be used to allow the PCR products to be loaded directly onto the gel. For this purpose biotechrabbit offers Direct-Load Master Mixes.
• When performing agarose gel electrophoresis, it is advisable to load different volumes (2–10 µl) of the PCR product if possible.
• Loading insufficient reaction volumes might give the impression no PCR product was amplified. Excessive PCR volume might cause difficulty in sizing and quantification.