Working with PCR

• General Considerations
  • Setting up the laboratory to avoid contamination
  • Special equipment
  • PCR reaction components
  • Thermal cycling profile for standard PCR
  • Properties of PCR enzymes

• Factors to Consider in RT-PCR
  • Choice of RT-PCR enzymes
  • Choice of reverse transcription primers
  • Preparation of template RNA
  • Primer design
  • RT-PCR procedures

• Prevention of Carryover Contamination
  • Preventing carryover contamination with uracil-DNA glycosylase
    
  • Preventing carryover contamination in RT-PCR


• Troubleshooting
  • No product
  • Misincorporation or low fidelity
  • Nonspecific bands
  • Smeared bands
  • Low yield

General Considerations

Setting up the Laboratory to Avoid Contamination

The ability of the polymerase chain reaction to amplify a single molecule means that trace amounts of DNA contaminants could serve as templates, resulting in amplification of the wrong template (false positives). Consider the points mentioned below to avoid PCR contamination from sources such as:

• Laboratory benches, equipment, and pipetting devices, which can be contaminated by previous DNA preparations, by plasmid DNA, or by purified restriction fragments.
  
• Cross-contamination between samples.
  
• Products from previous PCR amplifications.
  

Laboratory Facilities

• At a minimum, use physically separated working places for:
  • template preparation before PCR
  • setting up PCR reactions
  • post-PCR analysis
• Use thin-walled PCR tubes which are DNase- and RNase-free.
• Use special aerosol-resistant pipette tips and a dedicated (used only for PCR) set of pipettes, preferably positive displacement pipettes.
• If possible, set up PCR reactions under a fume hood that is equipped with UV light. Store a microcentrifuge and disposable gloves which are used only for PCR under the fume hood.
  

Sample Handling

• Use sterile techniques and always wear fresh gloves when working in the PCR area. Change gloves frequently, especially if you suspect they have become contaminated with solutions containing template DNA.
• Always use new and/or sterilized glassware, plasticware, and pipettes to prepare PCR reagents and template DNA.
• Autoclave all reagents and solutions that can be autoclaved without affecting their performance. Of course, primers, dNTPs, and Taq DNA Polymerase should not be autoclaved. Have your own set of PCR reagents and solutions that are used only for PCR. Store these reagents in small aliquots.
• When pipetting DNA, avoid creating aerosols that could carry contaminants.
• Always include control reactions, for example a negative ("no DNA") control which contains all reaction components except the template DNA, and a positive control that has been successfully used in previous PCRs.

Special Equipment

Type of Thermal Cycler

A thermal cycler must, at a minimum, accurately and reproducibly maintain the three PCR incubation temperatures (see Thermal Cycling Profile for Standard PCR), change from one temperature to another ("ramp") over a definable time, arrive at the selected temperatures without significant over- or undershoot, and cycle between the temperatures repeatedly and reproducibly.

Note: Cycling conditions have to be adjusted depending on the respective thermal cycler or primer/template combinations.

Type of Reaction Tubes

The reaction tubes affect the rate at which heat transfers from the thermal cycler to the reaction mixture. Therefore, preferably use thin-walled reaction tubes that are designed for PCR and that fit precisely into the wells of the particular brand of thermal cycler you are using.

PCR Reaction Components

Template

The quality of the template influences the outcome of the PCR. For instance, large amounts of RNA in a DNA template can chelate Mg²⁺ and reduce the yield of the PCR. Also, impure templates may contain polymerase inhibitors that decrease the efficiency of the reaction.

Note: To get the purest template, always use a purification product specifically designed to purify DNA, such as the High Pure PCR Template Preparation Kit.

The integrity of the template is also important. Template DNA should be of high molecular weight. To check the size and quality of the DNA, run an aliquot on an agarose gel. When testing a new template, always include a positive control with primers that amplify a product of known size and produce a good yield.

The amount of template in a reaction strongly influences performance in PCR. The recommended amount of template for standard PCR is:

• A maximum of 500 ng of human genomic DNA
• 1 – 10 ng bacterial DNA
• 0.1 – 1 ng plasmid DNA

Low amounts of template, for example, <10 ng human genomic DNA, will require specific reaction modifications, such as changes in cycle number, redesign of primers, use of Hot Start, etc.

Primers

In most PCR applications, it is the sequence and the concentration of the primers that determine the overall assay success. For convenience, several primer design software programs are available. These can be used to ensure that the primer sequences have the following general characteristics:

• 18 – 24 bases long
• No internal secondary structure
• 40 – 60% G/C
• Balanced distribution of G/C and A/T rich domains.
• Not complementary to each other at the 3´ ends (primer-dimers will not form).
• Melting temperature (Tm) that allows annealing temperatures of +55 to +65 °C (for maximum specificity use temperatures of +62 to +65 °C).

Note: Optimal annealing temperatures are often higher than the Tm of the primers (approximately +5 to +10 °C) and have to be determined empirically. For both primers, the Tm should be similar.

Bases that do not hybridize to the template may be added at the 5´ end of a primer (e.g., for introducing restriction sites into the amplification product). Primer concentrations between 0.1 and 0.6 µM are generally optimal. Higher primer concentrations may promote mispriming and accumulation of nonspecific product. Lower primer concentrations may be exhausted before the reaction is completed, resulting in lower yields of desired product.

Note: For some systems, a higher primer concentration (up to 1 µM) may improve results. When testing new primers, always include a positive control reaction with a template that has been tested for function in PCR. This control shows whether the primers are working. The Human Genomic DNA from Roche is a good control template for evaluation of human primer sequences.

Choice of DNA Polymerase

The choice of a DNA polymerase can profoundly affect the outcome of PCR. For most routine PCR, Taq DNA Polymerase has long been the standard PCR enzyme. However, Taq DNA Polymerase has its limitations. For most assays, the optimum amount of thermostable DNA polymerase (or a blend of polymerases) will be between 0.5 and 2.5 units / 50 µL reaction volume. Increased enzyme concentrations sometimes lead to decreased specificity.

MgCl₂ Concentration

Mg²⁺ forms soluble complexes with dNTPs to produce the actual substrate that the polymerase recognizes. The concentration of free Mg²⁺ depends on the concentrations of compounds that bind the ion, including dNTP, free pyrophosphate (PPi) and EDTA. For best results, always determine the optimal Mg²⁺ concentration empirically. The optimal Mg²⁺ concentration may vary from approximately 1 - 5 mM. The most commonly used Mg²⁺ concentration is 1.5 mM (with dNTPs at a concentration of 200 µM each). Mg²⁺ influences enzyme activity and increases the Tm of double-stranded DNA. Excess Mg²⁺ in the reaction can increase nonspecific primer binding and increase the nonspecific background of the reaction.

Deoxynucleotide Triphosphate (dNTP) Concentration

Always use balanced solutions of all four dNTPs to minimize polymerase error rate. Imbalanced dNTP mixtures will reduce Taq DNA Polymerase fidelity.

Note: For maximum convenience, a premixed, balanced mixture of dNTPs such as the PCR Nucleotide Mix may be added to the reaction mixture as a single reagent. In addition, PCR grade dATP, dGTP, dCTP, dTTP and a Deoxynucleoside Triphosphate Set, PCR Grade are available.

If you increase the concentration of dNTPs, you must also increase the Mg²⁺ concentration. Increases in dNTP concentration reduce free Mg²⁺, thus interfering with polymerase activity and decreasing primer annealing. For prevention of carryover contamination, a higher concentration of dUTP is usually used in place of dTTP (for details, see Preventing carryover contamination with uracil-DNA glycosylase). The final dNTP concentration should be 50 – 500 µM (each dNTP). The most commonly used dNTP concentration is 200 µM.

pH

Generally, the pH of the reaction buffer supplied with the corresponding thermostable DNA polymerase (pH 8.3 – 9.0) will give optimal results. However, for some systems, raising the pH may stabilize the template and enhance results.

Reaction Additives

In some cases, adding the following compounds can enhance the efficiency or specificity of PCR:

• Betaine (0.5 – 2 M)
• Bovine serum albumin (BSA; 100 ng/50 µL)
• Detergents
• Dimethylsulfoxide (DMSO; 2 – 10%) (v/v)
• Gelatine
• Glycerol (1 – 5%) (v/v)
• Pyrophosphatase (0.001 – 0.1 units/reaction)
• Spermidine
• T4 Gene 32 protein
• Formamide

Thermal Cycling profile for Standard PCR

Initial Denaturation

It is essential to denature the template DNA completely. Initial heating of the PCR mixture for 2 minutes at +94 to +95 °C is enough to completely denature complex genomic DNA so that the primers can anneal to the template as the reaction mix is cooled. If the template DNA is only partially denatured, it will tend to "snap-back" very quickly, preventing efficient primer annealing and extension, or leading to "self-priming", which can lead to false-positive results.

Denaturation Step During Cycling

Denaturation at +94 to +95 °C for 20 – 30 seconds is usually sufficient, but this must be adapted for the thermal cycler and tubes being used. For example, longer times are required for denaturation in 500 µL tubes than in 200 µL tubes. If the denaturation temperature is too low, the incompletely melted DNA "snap-backs" as described earlier, thus giving no access to the primers. Use a longer denaturation time or higher denaturing temperature for GC-rich template DNA.

Note: Never use a longer denaturation time than absolutely required for complete denaturation of template DNA. Unnecessarily long denaturation times decrease the activity of Taq DNA Polymerase.

Primer Annealing

For most purposes, annealing temperature has to be optimized empirically. The choice of the primer annealing temperature is probably the most critical factor in designing a high specificity PCR. If the temperature is too high, no annealing occurs, but if it is too low, non-specific annealing will increase dramatically. Primer-dimers will form if the primers have one or more complementary bases so that base pairing between the 3´ ends of the two primers can occur.

Primer Extension

For fragments up to 3 kb, primer extension is normally carried out at +72 °C. Taq DNA Polymerase can add approximately 60 bases per second at +72 °C. A 45-second extension is sufficient for fragments up to 1 kb. For extension of fragments up to 3 kb, allow about 45 seconds per kb. However, these times may need to be adjusted for specific templates. For improved yield, use the cycle extension feature of the thermal cycler. For instance, perform the first 10 cycles at a constant extension time (e.g., 45 seconds for a 1 kb product). Then, for the next 20 cycles, increase the extension time by 2–5 seconds per cycle (e.g., 50 seconds for cycle 11, 55 seconds for cycle 12, etc.). Cycle extension allows the enzyme more time to do its job, because as PCR progresses, there is more template to amplify and less enzyme (due to denaturation during the prolonged high PCR temperatures) to do the extension.

Cycle Number

In an optimal reaction, less than 10 template molecules can be amplified in less than 40 cycles to a product that is easily detectable on a gel stained with ethidium bromide. Most PCRs should, therefore, include only 25 to 35 cycles. As cycle number increases, nonspecific products can accumulate (Figure 1).

Figure 1. Effect of excessive cycling on impure and pure templates. A PCR product (245 bp amplicon from exon 6 of the dopamine 2 receptor gene) was reamplified in a series of reactions. In one set of experiments, the template was not purified before it was used. In the second set, the template was purified by agarose gel electrophoresis before reamplification. In both sets, the template was amplified for either 40, 60, or 72 cycles. Aliquots (8 µL) of the products were analyzed on a 3% agarose gel.

MWM: Molecular Weight Marker
40, 60, and 72: Number of amplification cycles.

Result: In both sets, the lowest number of cycles (40) produced the most specific product. In both the 60 and 72 cycle amplifications, a smear appeared which contained multimeric "specific" PCR products.
Photo courtesy of U. Finckh and A. Rolfs, Free University of Berlin, Germany.

Final extension

Usually, after the last cycle, the reaction tubes are held at +72 °C for 5 – 15 minutes to promote completion of partial extension products and annealing of single-stranded complementary products.

Properties of PCR enzymes

Table 1 shows the application profile of PCR enzymes provided by Roche.

Table 1 Application profile of PCR enzymes provided by Roche.

Length	The length of the sequences which can be amplified.
Specificity	The extent of nonspecific amplification in particular, if difficult templates are to be amplified. The higher the specificity, the lesser by-products are generated.
Reproducibility	Consistency of results obtained with different enzyme lots.
Sensitivity	Dependence of amplification efficiency on the amount of template. If the template is limited, an enzyme system with a high sensitivity should be used.
Robustness	Vulnerability of an enzyme system to contaminating agents or other factors which reduce the amplification efficiency, such as strong secondary structures.
Accuracy	Error rate of an enzyme system as defined by the relative number of misincorporated nucleotides compared to Taq DNA Polymerase.
Carryover Prevention	Defines whether an enzyme system can be used with dUTP instead of dTTP to apply procedures for PCR carryover prevention using uracil DNA glycosylase (UNG).

Factors to Consider in RT-PCR

Choice of RT-PCR Enzymes

Obviously, in RT-PCR, a major factor to consider is the choice of reverse transcriptase used to synthesize cDNA. Since each of the available enzymes has different enzymatic properties, one may be more suitable for a specific experiment than the others. The most important enzyme properties are discussed below.

Temperature Optima

Higher incubation temperatures can help eliminate problems of template secondary structure. In addition, high temperature improves the specificity of reverse transcription by decreasing false priming. Thus, thermoactive reverse transcriptases that can be incubated at high temperatures (+50 to 70 °C) are more likely to produce accurate copies of mRNA, especially if the template has a high GC content.

Note: At these high temperatures, use only specific primers; do not use oligo(dT) or random hexamer primers.

Divalent Ion Requirement

Most reverse transcriptases require a divalent ion for activity. Enzymes that use Mg²⁺ are likely to produce more accurate cDNA copies than those that use Mn²⁺, since Mn²⁺ adversely affects the fidelity of DNA synthesis.

Specificity and Sensitivity

Reverse transcriptases have differing ability to copy small amounts of template (sensitivity). They also differ in their ability to transcribe RNA secondary structures accurately (specificity).

Enzyme Profiles

Table 2 shows the application profiles of RT-PCR enzymes and kits provided by Roche.

Table 2. Application profiles of RT-PCR enzymes and kits provided by Roche.

Choice of Reverse Transcription Primers

Priming affects the size and specificity of the cDNA produced. There are three types of primers that may be used for reverse transcription:

• Oligo(dT)12–18, which binds to the endogenous poly(A)+ tail at the 3´ end of mammalian mRNA. This primer often produces a full-length cDNA.
• Random hexanucleotides, which bind to mRNA at a variety of complementary sites and lead to partial length (short) cDNAs. Random hexanucleotides may be ideal for overcoming the difficulties presented by extensive secondary structure in the template. These primers may also transcribe more efficiently 5´ regions of the mRNA.
• Specific oligonucleotide primers, which selectively prime the mRNA of interest. This type of primer has been used very successfully in diagnostic assays.

Preparation of template RNA

Successful RT-PCR requires a high quality, intact RNA template. Use the following guidelines to help prepare this template:

• To minimize the activity of RNases that are released during cell lysis, include Protector RNase Inhibitor in the lysis mix or use methods that simultaneously disrupt cells and inactivate RNases.
• Take steps to eliminate all potential sources of RNase contamination from glassware, plasticware, reagents, etc.
• Use a product specifically designed for nucleic acid purification to prepare starting template RNA, such as the RNA preparation products provided by Roche.
• Use purified mRNA as template, rather than total RNA. Starting with poly(A)+ mRNA will greatly increase the likelihood of successful amplification of rare mRNAs, since the proportion of mRNA in a total RNA preparation is quite low (typically, 1 – 5% of total RNA from a mammalian cell).
• If using mRNA as template, check the integrity of the mRNA by gel electrophoresis before using it in RT-PCR. The mRNA should appear as a smear between approximately 500 bp and 8 kb. Most of the mRNA should be between 1.5 kb and 2 kb.

Primer design

RT-PCR amplification of a particular mRNA sequence requires two PCR primers that are specific for that mRNA sequence. The primer design should also allow differentiation between the amplified product of cDNA and an amplified product derived from contaminating genomic DNA. There are two approaches to designing the required primers (Figure 2).

Figure 2. Primer design approaches.

Panel 1: Make primers that anneal to sequences in exons on both sides of an intron. With these primers, any product amplified from genomic DNA will be much larger than a product amplified from intronless mRNA.
Panel 2: Make primers that span exon/exon boundaries on the mRNA. Such primers should not amplify genomic DNA.

RT-PCR Procedures

RT-PCR can be performed as either a two-step or an one-step procedure. Each has certain advantages.

1. Two-step procedures

A. Two tube, two-step procedure

In the first tube, first-strand cDNA synthesis is performed under optimal conditions, using either random hexamers, oligo(dT) primers (generating a cDNA pool), or sequence-specific primers. An aliquot of the RT reaction is then transferred to another tube (containing thermostable DNA polymerase, DNA polymerase buffer, and PCR primers) for PCR.

Advantages of this approach: This method is useful for experiments where multiple transcripts have to be analyzed from the same RT reaction or for specific applications, such as Differential Display Reverse Transcription (DDRT) or Rapid Amplification of cDNA Ends (RACE). Also, since the RT reaction is performed under optimal conditions, this approach produces the longest RT-PCR products (up to 14 kb in length, if the appropriate enzymes are used).

B. One tube, two-step procedure

In the first step, reverse transcriptase produces first-strand cDNA in the presence of Mg²⁺ ions, high concentrations of dNTPs, and either specific or nonspecific [oligo(dT)] primers (reaction volume, 20 µL). Following the RT reaction, an optimized PCR buffer (without Mg²⁺ ions), a thermostable DNA polymerase, and specific primers are added to the tube and PCR is performed. This approach may be useful when template amounts are limited, since the entire RT reaction is used in the subsequent PCR.

A two-step procedure has the following advantages:

• Optimizes reaction conditions. The two-step format allows both reverse transcription and PCR to be performed under optimal conditions to ensure efficient and accurate amplification.
• Provides flexibility. Two-step procedures allow the product of a single cDNA synthesis reaction to be used for analysis of multiple transcripts. This flexibility is valuable for such specialized applications as rapid amplification of cDNA ends (RACE) and Differential Display Reverse Transcription (DDRT).
• Amplifies long sequences. With the right combination of reverse transcriptase and thermostable DNA polymerase, two-step RT-PCR can amplify RNA sequences up to 14 kb long.

2. One tube, one-step procedure (coupled RT-PCR)

Both cDNA synthesis and PCR amplification are performed with the same buffer and site-specific primers, eliminating the need to open the reaction tube between the RT and PCR steps. In addition to the higher sensitivity of this approach (as in the one tube, two-step reaction above), the one-step approach minimizes the chance of contamination, since the entire reaction is performed with minimal pipetting steps and without opening the tube. In addition, this approach permits direct analysis of a specific transcript, since the primers used in both steps are sequence-specific. Finally, the thermoactive reverse transcriptase used in this procedure allow a high RT reaction temperature (+50 to +72 °C), which reduces false priming and increases the specificity of the reaction by eliminating secondary mRNA structure.

A one-step procedure has the following advantages:

• Minimizes time required. The one-step reaction has fewer pipetting steps than the two-step reaction, significantly reducing the time needed to perform RT-PCR and eliminating pipetting errors.
• Reduces the risk of contamination. The entire one-step reaction takes place in a single tube, with no transfers required and no need to open the reaction tube (steps where contamination of the PCR sample can take place).
• Improves the sensitivity and specificity of cDNA synthesis. Two characteristics of the one-step reaction provide increased yield and efficiency: (1) the cDNA reaction is performed at a high temperature (to eliminate problems with RNA secondary structure), and (2) the entire cDNA sample is used as template for PCR.

Prevention of Carryover Contamination

Preventing Carryover Contamination with Uracil-DNA Glycosylase

The polymerase chain reaction (PCR) can amplify a single molecule over a billionfold. Thus, even minuscule amounts of a contaminant can be amplified and lead to a false-positive result. Such contaminants are often products from previous PCR amplifications (carryover contamination). Therefore, researchers have developed methods to avoid such contamination.

One common strategy is substituting dUTP for dTTP during PCR amplification, to produce uracil-containing DNA (U-DNA). Treating subsequent PCR reaction mixtures with Uracil-DNA Glycosylase (UNG) prior to PCR amplification and subsequent cleavage of apyrimidinic polynucleotides at elevated temperature (+95 °C) under alkaline conditions (during the initial denaturation step) will remove contaminating U-DNA from the sample (Figure 3). This method, of course, requires that all PCR reactions in the lab be carried out with dUTP instead of dTTP.

Figure 3. Removal of contaminating U-DNA with uracil-DNA glycosylase.

Note the following when using dU-containing PCR products in downstream applications:

• PCR products containing dU perform as well as those containing dT when used as hybridization targets or as templates for dideoxy sequencing.
• PCR products containing dU can be cloned directly, if they are transformed into ung–bacterial hosts.
• A dU-containing substrate is readily digested by some common restriction enzymes (e.g., Eco RI and Bam HI), while others show reduced activity (e.g., Hpa I, Hind II, Hind III) on these substrates.
• We do not recommend the use of dU-containing DNA for protein binding or DNA-protein interaction studies.

Preventing Carryover Contamination in RT-PCR

There are two ways of preventing carryover contamination when amplifying RNA:

• Use a one-tube, one-step RT-PCR procedure to minimize the number of handling and pipetting steps and the number of times that the reaction tube must be opened. This minimizes the possibility of carryover contamination in the RT-PCR itself.
• Perform one-step RT-PCR in the presence of dUTP, so that all products will contain dU and can be removed from subsequent PCR with UNG (see Preventing Carryover Contamination with Uracil-DNA Glycosylase). This prevents carryover contamination in subsequent PCRs.

Troubleshooting

Here are some troubleshooting hints that we have gathered regarding PCR, based on the five most common symptoms observed. Before you begin, please make sure that you have reviewed the other application hints.

No Product

Here are some troubleshooting hints that we have gathered if your PCR yields no product.

1. Non-optimal Mg²⁺ concentration

Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.

2. The amount of template in the reaction is not optimal

The necessary amount of template varies from reaction to reaction. As a guideline, use 100 - 750 ng human DNA (10⁵ - 10⁶ copies) per 100 µL reaction. The amount of enzyme should be optimized for each template.

Suggestion:

• Titrate the amount of template in the reaction.
• Perform optimization experiment varying enzyme concentration by 0.50 increments in suggested range (0.5 to 5.0 units).

3. An enzyme inhibitor is present in the reaction

Known inhibitors of PCR include:

• 50 mM ammonium chloride
• EDTA (metal chelator)
• >0.8 µM hematin
• PBS (phosphate will bind free magnesium)
• >0.02 % sarcosyl
• 0.5 M urea
• >5% DMF
• >10% formamide
• Heparin
• >20% PEG deoxycholate
• >0.01% SDS (can be reversed with equal molar ratio of NP40 and Tween 20)
• >10% DMSO
• >20% glycerol
• >0.4% N-octylglucoside
• Residual phenol
• >0.06% sodium deoxycholate
• Pyrophosphate

Suggestion: Reduce or remove the concentration of any inhibitor in the reaction mixture.

4. Primer annealing temperature is too high or too low

Primer annealing temperature is typically +50 to +60 °C (may be higher or lower based on primer sequence and buffer components).

Suggestion: Determine Tm/annealing temperature based on the following equations:

If primers are 20-35 bases
Tp = 22 + 1.46 (Ln)
Ln = 2(# G or C) + (# A or T)
Tp = Effective annealing temperature ± 2 - 5

If primers are 14 - 70 bases
Tm = 81.5 + 16.6 (log10 [J+]) + 0.41 (% G + C) - (600/l) - 0.063 (% Formamide) + 3 to 12
[J+] = concentration of monovalent cations
l = length of oligo

5. Primers are degraded or not optimal

Primers should have the same number A and T's versus G and C's, and they should be at least 14 bases for specificity.

Suggestions:

• If primers are short and A-T rich, add 0.9 - 2.0% (v/v) DMSO.
• If primers are G-C rich, add 1 - 10% (v/v) formamide.
• Double-check priming sequence, use primer design program if available.
• Check aliquot of primers on a gel to ensure they are not degraded.

6. Incomplete template denaturation

Insufficient heating during the denaturation step is a common cause of failure in a PCR reaction. It is very important that the reaction reaches a temperature at which complete strand separation occurs. A temperature of about +94 °C for 2 minutes should be adequate in most cases.

As soon as the sample reaches +94 °C, it can be cooled to the annealing temperature. Extensive denaturation is probably unnecessary and limited exposure to elevated temperatures helps maintain maximum polymerase activity throughout the reaction.

DNA reaction buffers with higher Mg concentration (4 - 5 mM) may require a higher denaturation temperature to allow complete separation of the DNA template strands. It is recommended to use the supplied buffer without adding further magnesium.

Suggestions:

• Increase initial denaturation temperature to +95 to +97 °C.
• Denature DNA minus enzyme and buffer for 4 - 6 minutes.
• Increase cycling denaturation time 15 - 30 seconds.
• Try Hot Start technique.
• Closed circular DNA should be cleaved before the PCR, uncut circles renaturate too fast.

7. Machine-based error

Suggestions:

• Calibrate the heating block and confirm actual block temperature.
• Run a diagnostic program for the particular machine - contact the machine manufacturer for specifics.

8. Mispriming caused by secondary structure of template, snapback, or excessive homology at 3´ ends of primers

Suggestions:

• Increase initial denaturation temperature to +95 to +97 °C.
• Denature DNA minus enzyme and buffer for 4 - 6 minutes.
• Increase cycling denaturation time 15 - 30 seconds.
• Try Hot Start technique.
• Add T4 Gene 32 protein 3 - 5 µL / mL.
• When designing primers, make sure there is no more than 2 bases of homology at the 3´ end. Use a primer design program if available.
• Consider addition of cosolvent to reaction buffer:
  • 3 - 15% DMSO
  • 1 - 10% formamide
  • 5 - 15% polyethylene glycol
  • 10 - 15% glycerol

9. NaCl concentration above 50 mM

Suggestions: Reduce NaCl concentration

10. KCl concentration above 50 mM

Suggestions: Reduce KCl concentration

Misincorporation or Low Fidelity

Here are some troubleshooting hints that we have gathered regarding misincorporation or low fidelity of PCR reactions.

1. Non-optimal Mg²⁺ concentration

Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.

2. Nucleotide concentration is too high or unbalanced

The standard concentration is 20 - 200 µM of each nucleotide.

Suggestions:

• Check the concentration of stock solutions of all nucleotides.
• Double-check the final concentrations of all nucleotides.

3. The pH of the reaction buffer is too high

A pH of 8.3 is optimal.

Suggestions:

• pH of reaction buffer can be lowered to 5 - 6.
• Base substitution changes decreased by sixtyfold and frame-shift changes decreased by elevenfold with a 3 unit pH decrease.

4. Mispriming caused by secondary structure of template, snapback, or excessive homology at 3´ ends of primers

Suggestions:

• Increase initial denaturation temperature to +95 to +97 °C.
• Denature DNA minus enzyme and buffer for 4 - 6 minutes.
• Increase cycling denaturation time 15 - 30 seconds.
• Try Hot Start technique.
• Add T4 Gene 32 protein 3 - 5 µL / mL.
• When designing primers, make sure there is no more than 2 bases of homology at the 3´ end. Use a primer design program if available.
• Consider addition of cosolvent to reaction buffer:
  • 3 - 15% DMSO
  • 1 - 10% formamide
  • 5 - 15% polyethylene glycol
  • 10 - 15% glycerol

5. Damaged template DNA

Suggestions:

• Minimize cycle number, since repeated heating/cooling at high pH can damage template (most templates require 25 - 35 cycles).
• Check integrity of template DNA.
• Reduce cycling denature time to 15 - 30 seconds

Nonspecific Bands

Here are some troubleshooting hints that we have gathered regarding nonspecific bands after PCR reactions.

1. Non-optimal Mg²⁺ concentration

Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.

2. Nucleotide concentration is too high or unbalanced

The standard concentration is 20 - 200 µM of each nucleotide.

Suggestions:

• Check the concentration of stock solutions of all nucleotides.
• Double-check the final concentrations of all nucleotides.

3. DNA contamination/carryover

Suggestions:

• Test for carryover by performing PCR without adding target DNA.
• Avoid carryover (see below).

To prevent carryover, use good laboratory practices:

• Physically isolate PCR preps and products
• Autoclave solutions, tips, and tubes
• Aliquot reagents to minimize repeated sampling (no more than 20 reactions per aliquot)
• Eliminate aerosols by using positive-displacement pipettes
• Premix reagents
• Add DNA to reaction last
• Choose positive and negative controls carefully
• Soak gel box and combs in 1 M HCl to depurinate DNA
• Use new razor blades to excise bands
• Cover UV box with fresh plastic wrap
• Always use oil overlay

To eliminate contamination/carryover:

• UV irradiation: Mix all components, except template DNA; irradiate in clear 0.5 mL polypropylene tubes in direct contact with glass transilluminator (254 and 300 nm UV bulbs) for 5 minutes.
• UNG digestion: Incorporate dUTP nucleotides into reaction and do subsequent uracyl DNA glycosylase digestion.

4. Primer annealing temperature is too low

Primer annealing temperature is typically +50 to +60 °C (may be higher or lower based on primer sequence and buffer components).

Suggestion: Determine Tm/annealing temperature based on the following equations:

If primers are 20-35 bases
Tp = 22 + 1.46 (Ln)
Ln = 2(# G or C) + (# A or T)
Tp = Effective annealing temperature ± 2 - 5

If primers are 14 - 70 bases
Tm = 81.5 + 16.6 (log10 [J+]) + 0.41 (% G + C) - (600/l) - 0.063 (% Formamide) + 3 to 12
[J+] = concentration of monovalent cations
l = length of oligo

5. Mispriming caused by secondary structure of template, snapback, or excessive homology at 3´ ends of primers

Suggestions:

• Increase initial denaturation temperature to +95 to +97 °C.
• Denature DNA minus enzyme and buffer for 4 - 6 minutes.
• Increase cycling denaturation time 15 - 30 seconds.
• Try Hot Start technique.
• Add T4 Gene 32 protein 3 - 5 µL / mL.
• When designing primers, make sure there is no more than 2 bases of homology at the 3´ end. Use a primer design program if available.
• Consider addition of cosolvent to reaction buffer:
  • 3 - 15% DMSO
  • 1 - 10% formamide
  • 5 - 15% polyethylene glycol
  • 10 - 15% glycerol

6. Primers are degraded or not optimal

Primers should have the same number A and T's versus G and C's, and they should be at least 14 bases for specificity.

Suggestions:

• If primers are short and A-T rich, add 0.9 - 2.0% (v/v) DMSO.
• If primers are G-C rich, add 1 - 10% (v/v) formamide.
• Double-check priming sequence, use primer design program if available.
• Check aliquot of primers on a gel to ensure they are not degraded.

7. Primer concentration is too high

Suggestion: Adjust the primer concentration (0.1 - 1.0 µM of each primer is optimal).

8. Cycle number is too high

Most templates require 25 - 30 cycles.

Suggestion: Cycle number should be based on starting concentration of template DNA.

If the number of target molecules in your sample is...	Then we recommend the following number of cycles...
3 x 105	25 - 30
1.5 x 104	30 - 35
1.0 x 103	35 - 40
50	40 - 45

9. Incorrect template to enzyme ratio

The necessary amount of template varies from reaction to reaction. As a guideline, use 100 - 750 ng human DNA (10⁵ - 10⁶ copies) per 100 µL reaction. The amount of enzyme should be optimized for each template.

Suggestions:

• Titrate the amount of template in the reaction.
• Perform optimization experiment varying enzyme concentration by 0.50 increments in suggested range (0.5 to 5.0 units).

Smeared Bands

Here are some troubleshooting hints that we have gathered regarding smeared bands after PCR reactions.

1. Non-optimal Mg²⁺ concentration

Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.

2. Nucleotide concentration is too high or unbalanced

The standard concentration is 20 - 200 µM of each nucleotide.

Suggestions:

• Check the concentration of stock solutions of all nucleotides.
• Double-check the final concentrations of all nucleotides.

3. DNA contamination/carryover

Suggestions:

• Test for carryover by performing PCR without adding target DNA.
• Avoid carryover (see below).

To prevent carryover, use good laboratory practices:

• Physically isolate PCR preps and products
• Autoclave solutions, tips, and tubes
• Aliquot reagents to minimize repeated sampling (no more than 20 reactions per aliquot)
• Eliminate aerosols by using positive-displacement pipettes
• Premix reagents
• Add DNA to reaction last
• Choose positive and negative controls carefully
• Soak gel box and combs in 1 M HCl to depurinate DNA
• Use new razor blades to excise bands
• Cover UV box with fresh plastic wrap
• Always use oil overlay

To eliminate contamination/carryover:

• UV irradiation: Mix all components, except template DNA; irradiate in clear 0.5 mL polypropylene tubes in direct contact with glass transilluminator (254 and 300 nm UV bulbs) for 5 minutes.
• UNG Digestion: Incorporate dUTP nucleotides into reaction and do subsequent uracyl DNA glycosylase digestion.

4. Primer annealing temperature is too low

Primer annealing temperature is typically +50 to +60 °C (may be higher or lower based on primer sequence and buffer components).

Suggestion: Determine Tm/annealing temperature based on the following equations:

If primers are 20-35 bases
Tp = 22 + 1.46 (Ln)
Ln = 2(# G or C) + (# A or T)
Tp = Effective annealing temperature ± 2 - 5

If primers are 14 - 70 bases
Tm = 81.5 + 16.6 (log10 [J+]) + 0.41 (% G + C) - (600/l) - 0.063 (% Formamide) + 3 to 12
[J+] = concentration of monovalent cations
l = length of oligo

5. Mispriming caused by secondary structure of template, snapback, or excessive homology at 3´ ends of primers

Suggestions:

• Increase initial denaturation temperature to +95 to +97 °C.
• Denature DNA minus enzyme and buffer for 4 - 6 minutes.
• Increase cycling denaturation time 15 - 30 seconds.
• Try Hot Start technique.
• Add T4 Gene 32 protein 3 - 5 µL / mL.
• When designing primers, make sure there is no more than 2 bases of homology at the 3´ end. Use a primer design program if available.
• Consider addition of cosolvent to reaction buffer:
  • 3 - 15% DMSO
  • 1 - 10% formamide
  • 5 - 15% polyethylene glycol
  • 10 - 15% glycerol

6. DNase activity (indicated by smears visible on gel below expected band size)

Suggestions:

• Make fresh stock solutions.
• Check the integrity of template DNA.

7. Oil contamination of gel sample

Primer annealing temperature is typically +50 to +60 °C (may be higher or lower based on primer sequence and buffer components).

Suggestion:Spin the reaction tube and carefully extract the oil layer from the surface.

8. Incorrect template to enzyme ratio

The necessary amount of template varies from reaction to reaction. As a guideline, use 100 - 750 ng human DNA (10⁵ - 10⁶ copies) per 100 µL reaction. The amount of enzyme should be optimized for each template.

Suggestions:

• Titrate the amount of template in the reaction.
• Perform optimization experiment varying enzyme concentration by 0.50 increments in suggested range (0.5 to 5.0 units).

Low Yield

Here are some troubleshooting hints that we have gathered regarding low yield after PCR reactions.

1. Primer annealing temperature is too high

Primer annealing temperature is typically +50 to +60 °C (may be higher or lower based on primer sequence and buffer components).

Suggestion: Determine Tm/annealing temperature based on the following equations:

If primers are 20-35 bases
Tp = 22 + 1.46 (Ln)
Ln = 2(# G or C) + (# A or T)
Tp = Effective annealing temperature ± 2 - 5

If primers are 14 - 70 bases
Tm = 81.5 + 16.6 (log10 [J+]) + 0.41 (% G + C) - (600/l) - 0.063 (% Formamide) + 3 to 12
[J+] = concentration of monovalent cations
l = length of oligo

2. Template not clean or degraded

For example, protease contamination can degrade the polymerase.

Suggestions:

• Try Hot Start technique.
• Purify the DNA template to the highest degree possible, including proteinase K digest.

3. An enzyme inhibitor is present in the reaction

Known inhibitors of PCR include:

• 50 mM ammonium chloride
• EDTA (metal chelator)
• >0.8 µM hematin
• PBS (phosphate will bind free magnesium)
• >0.02 % sarcosyl
• 0.5 M urea
• >5% DMF
• >10% formamide
• Heparin
• >20% PEG deoxycholate
• >0.01% SDS (can be reversed with equal molar ratio of NP40 and Tween 20)
• >10% DMSO
• >20% glycerol
• >0.4% N-octylglucoside
• Residual phenol
• >0.06% sodium deoxycholate
• Pyrophosphate

Suggestion: Reduce or remove the concentration of any inhibitor in the reaction mixture.

4. Not enough template in the reaction

The necessary amount of template varies from reaction to reaction. As a guideline, use 100 - 750 ng human DNA (10⁵ - 10⁶ copies) per 100 µL reaction. The amount of enzyme should be optimized for each template.

Suggestions:

• Titrate the amount of template in the reaction.
• Perform optimization experiment varying enzyme concentration by 0.50 increments in suggested range (0.5 to 5.0 units).

5. Extension temperature too high

Optimal extension temperature and time depends on fragment size:

• +72 °C for 20 seconds for fragments <500 bp.
• +72 °C for 40 seconds for fragments 1.2 bp.

Suggestions: Longer times, not higher temperatures should be used when longer templates or suspected secondary structure is present.

6. Enzyme activity is low

For Roche polymerases, 100% activity is guaranteed through the control date.

Suggestions:

• Check the control date. If necessary, obtain fresh enzyme.
• Try Hot Start technique to help retain activity through thermal cycling.

7. Cycle number is too high

Most templates require 25 - 30 cycles.

Suggestion: Cycle number should be based on starting concentration of template DNA.

If the number of target molecules in your sample is...	Then we recommend the following number of cycles...
3 x 105	25 - 30
1.5 x 104	30 - 35
1.0 x 103	35 - 40
50	40 - 45

8. Nucleotides hydrolyzed

Always store nucleotide stock solutions at a concentration of at least 10 mM, 100 mM is best. Significant hydrolysis occurs after storing at 1 mM for 2 months.

Dissolve NTPs or dNTPs in water at an expected concentration of 10 mM. Using 0.05 M Tris-base and pH paper, adjust the pH to 7.0. Dilute an aliquot of the neutralized NTP or dNTP appropriately and read the optical density at the wavelengths given in the following table. Calculate the actual concentration using the values for the extinction coefficients. Freeze away in small aliquots at -20 °C.

Base	Wavelength	Extinction coefficients for bases e (M-1 cm-1)
A	259	1.54 x 104
T	260	7.4 x 103
G	253	1.37 x 104
C	271	9.1 x 103
U	262	1.0 x 104

The lithium and sodium salts have equivalent stability and work equally well in PCR, sequencing, and labeling applications. Lithium salts are more soluble in ethanol than sodium salts. Thus, removal of lithium salts by ethanol precipitation is more efficient than removal of sodium salts. Using lithium salt nucleotide preparations reduces salt-induced artifacts and increases the legibility of sequencing gels.

9. Nucleotide concentration is too high or unbalanced

The standard concentration is 20 - 200 µM of each nucleotide.

Suggestions:

• Check the concentration of stock solutions of all nucleotides.
• Double-check the final concentrations of all nucleotides.

10. Primer concentration is too low

Suggestion: Adjust the primer concentration (0.1 - 1.0 µM of each primer is optimal).

11. Machine-based error

Suggestions:

• Calibrate the heating block and confirm actual block temperature.
• Run a diagnostic program for the particular machine - contact the machine manufacturer for specifics.

12. Plateau effect

Possible causes of the plateau effect/solutions:

• Utilization of dNTP's - dNTP concentration should be 20 - 200 µM each.
• End product pyrophosphate inhibition - reduce cycle number to 20 - 35 to reduce pyrophosphate formation.
• Incomplete denaturation of product at high concentration - use stepwise cycling, increasing time of denaturation in later cycles.
• Substrate excess in substrate/enzyme ratio - use stepwise cycling, increasing extension time in later cycles or enzyme concentration.

13. Evaporation

Evaporation can lead to higher concentrations of components, which may inhibit enzyme activity. The change in volume also leads to changes in the thermal profile inside the reaction tubes.

Suggestion: Use 100 µL mineral oil overlay/reaction.