Insufficient cycles

Not enough cycles may lead to insufficient amplification

• Generally start with 20 – 35 cycles; increase the number of cycles by 3-5 at a time until sufficient amplification is observed
• If the template concentration is low (< 1 µg/mL dsDNA), use more cycles; extend to 40 cycles if fewer than 10 copies of DNA are available

Denaturation temperature is too low or high

• Low temperature results in incomplete denaturation (double-stranded DNA may not denature)
• High temperatures may reduce enzyme activity

• Optimize the denaturation temperature
• Typically, a denaturation temperature of 94-98°C is used
• Use the temperature gradient function on your PCR machine to optimise the denaturation temperature in one PCR run. This function enables you to evaluate several different denaturation temperatures across a single block.

Denaturation time is too long or too short

• Too long: the DNA may be degraded and the enzyme activity may be reduced
• Too short: the DNA will not be completely denatured

• Optimize the denaturation time
• For initial denaturation, use 3-5 min
• For denaturation during cycling, use 10-30s

Annealing time is too short

The primers do not have enough time to bind to the template.

• Use an annealing time of at least 20-30s

Annealing temperature is too high

The primers cannot bind to the template.

•  Typically, use an annealing temperature that is 3-5°C lower than the T_m of the primers
• Use a thermal gradient to optimize the annealing temperature stepwise in 1-2°C increments, starting at 5oC below the T_m (of the lower T_m, if using pairs of primers)
• Adjust the annealing temperature if you are using a PCR additive or co-solvent
• Use a touch-down PCR protocol

Extension time is too short

There is insufficient time for complete replication of the target.

• Typical extension times are 30s-1 min/kb
•  Longer templates will need long extension times, or consider  using a faster enzyme such as our BlastTaq™ DNA Polymerase (Cat. No. G894) which is 3X faster than regular Taq
• Include a final extension time of at least 5 min

Extension time is too long

Overly long extension times can inactivate the PCR enzyme.

• Reduce the temperature by 3-4°C – this will help with the thermostability of the polymerase and keep it active, especially when amplifying longer targets (>10 kb)

Complex template

E.g. Template contains GC-rich or secondary structures.

GC content that is  >65% is typically difficult to amplify due to its increased stability and resistance to denaturation.

• Increase the annealing temperature – optimize using a thermal gradient
• Use high processivity or hot-start DNA polymerases such as our MegaFi™ Pro Fidelity DNA Polymerase (Cat. No. G886) or BlasTaq™ HotStart DNA Polymerase (Cat. No. G896) – these PCR enzymes have a high affinity for DNA templates and are better for amplifying difficult targets
• Use a PCR additive or co-solvent, such as DMSO or secondary structure destabilizer – this denatures GC-rich sequences and secondary structures; do not exceed 10% per reaction sample
•  Increase denaturation time and/or temperature – this helps primer binding and enzyme thermostability; especially useful for GC-rich templates
• Use a touch-down PCR protocol

Damaged or degraded template

The template may have been sheared or degraded.

•  If possible, use fresh template or re-make the template DNA (especially if it is genomic DNA as old stocks are prone to degradation or shearing)
• Minimize shearing and nicking of gDNA during isolation by minimizing vortexing and freeze-thawing
• Store DNA in TE buffer (pH 8.0) to prevent degradation by nucleases
• Before and after incubation with Mg²⁺, analyze DNA via gel electrophoresis to evaluate the integrity of the template, if necessary
• Limit or avoid UV exposure of DNA
• Use dyes with a longer-wavelength excitation to visualize DNA; irradiation from longer-wavelengths are less damaging to the DNA

Impure template

• May contain PCR inhibitors such as residual salts and other compounds that  inhibit DNA polymerases
• EDTA present in an RNA solution will chelate the Mg²⁺ in the buffer, which is normally present to enhance PCR enzyme activity

• If inhibitors are suspected, dilute the existing template or use a fresh template and increase the number of cycles
• Set up a control reaction using a pure plasmid and compare with pure plasmid spiked with your template to observe if inhibitors could be present in your template 
• Ensure that no residual PCR inhibitors such as phenol, EDTA, and Proteinase K are present following chemical or enzymatic DNA purification protocols
• Purify the starting template by alcohol precipitation, drop dialysis, or using a commercial clean up kit to remove residual salts or ions (K⁺, Na⁺, etc.)
• Use DNA polymerases with high processivity, which display high tolerance to common PCR inhibitors carried over from soil, blood, plant tissues, etc.
• If EDTA is present in an RNA solution, add more Mg²⁺ to compensate for the loss

Insufficient template in the reaction

If the template concentration is too low this can result in insufficient amplification.

• Increase the number of cycles in increments of 5 or increase the amount of template
• Use different concentrations of the template via a dilution series to optimize the input amount
•  Use DNA polymerases with high sensitivity of amplification such as our BlasTaq™ DNA Polymerase (Cat. No. G894)

Excess template

• May bind all the primers in early amplification cycles and result in insufficient primers for subsequent rounds of amplification
• Polymerase can be inhibited from inhibitor compounds carried over from the template
• Can cause inefficient denaturation

•  Use different concentrations of the template via a dilution series to optimize the input amount; typically a range of 100-200 ng of mammalian genomic DNA is used
• Increase the denaturation time and temperature
• Increase annealing temperature

Target is too long

PCR component concentrations and/or cycling conditions may be insufficient for longer targets.

• Increase duration of PCR steps, especially the extension time
• Check the amplification length capability of the polymerase used
• Use polymerases designed for long PCR such as our MegaFi™ Fidelity DNA Polymerase (Cat. No. G595) or BlasTaq™ HotStart DNA Polymerase (Cat. No.G896); choose DNA polymerases with high processivity
• Reduce the annealing and extension temperatures

Impure primers

Contaminants in primers may inhibit PCR.

• Use desalted primers or more highly purified primers
• Dilute primers to check for inhibitory effects

Primer concentration is too low

Low primer concentrations  may result in inefficient annealing.

•  Optimize concentration of primers; typical primer concentrations range from, 0.1-1 μM in the final reaction
• For long target PCR or PCR with degenerate primers use a minimum concentration of 0.5 µM
• Ensure primer concentrations are balanced to ensure even amplification of all DNA targets

Incorrect primer design/synthesis

Poor primer design will result in inefficient amplification of the DNA template.

• Primers should be a minimum of 18 bp long and have a minimum melting temperature of 52℃
• Review primer design and verify that the primers have the correct sequence, are specific to the target of interest, and are complementary to the template
• Check that primers are non-complementary internally or to each other as this could cause formation of primer-dimers (self-primers)
• Use a primer design program or check out our primer design service
• Avoid GC-rich 3’ ends and ensure that primers have less than 60% GC content – aim for a maximum of 45% GC content
• Avoid stretches of 4 or more G nucleotides in a row as this may cause problems during primer synthesis
• Try to design the primers to end with a G or C nucleotide on the 3’ end, as this “GC clamp” can help create a stronger bond for the amplification to initiate
• Perform a BLAST search to avoid amplification of pseudogenes or unintended regions
• When working with genomic DNA, check for introns between primer sites as if the introns are too long, it may result in incomplete product/no amplification
• Use nested primers

Old/degraded primers

Degraded primers will result in ineffective amplification of the DNA template.

• Aliquot primers after resuspending and store them properly
•  Use fresh primers/obtain new primers and avoid excessive freeze-thawing to reduce degradation risk
• You can confirm if primers are degraded using a polyacrylamide gel or HPLC methods
• Double-check your calculations for the optimal enzyme concentration - this will depend on the length and difficulty of the template and should follow manufacturer’s recommendations
• Choose DNA polymerases with high sensitivity for amplification such as our BlasTaq™ HotStart DNA Polymerase (Cat. No. G595)
• Add more enzyme if the reaction mix contains a high concentration of an additive (e.g.. DMSO or formamide) or other inhibitors from the sample source

Polymerase concentration is too low

If the concentration is too low, not all the PCR products will be fully replicated.

•  Double-check your calculations for the optimal enzyme concentration - this will depend on the length and difficulty of the template and should follow manufacturer’s recommendations
• Choose DNA polymerases with high sensitivity for amplification such as our BlasTaq™ HotStart DNA Polymerase (Cat. No. G595)
• Add more enzyme if the reaction mix contains a high concentration of an additive (e.g.. DMSO or formamide) or other inhibitors from the sample source

Inappropriate DNA polymerase

If standard Taq is not sufficient for your template, there are many speciality enzymes available to try.

• Use a different polymerase: try our BlasTaq™ HotStart DNA Polymerase (Cat. No. G595). This PCR enzyme:
  • Prevents primer degradation that can occur due to the 3’- 5’ exonuclease activity of proofreading DNA polymerases
  • Eliminates nonspecific amplification, which increases yield
• Setup PCR on ice if using non-hot-start DNA polymerases and add polymerase last to reaction mix to ensure amount is completely transferred and premature activation does not occur

Polymerase is inactive

Polymerase stocks can become inactivated due to excessive freeze-thawing.

• Run another PCR with fresh polymerase from a different batch or lot

Impure/degraded dNTPs

Contamination of the dNTP mix may inhibit PCR or result in incomplete or incorrect amplification
Degraded dNTPs can be caused by repeated freeze-thaw cycles

• Use high-quality dNTPs such as our dNTP mix
• Make a new dNTP solution
• Aliquot dNTPs to reduce degradation from excessive freeze-thaw cycles

dNTP concentration is too high or low

If the dNTP concentration is too high, Mg²⁺ depletion occurs and will have an inhibitory effect.

• Use a dNTP mix to prevent errors
• Aim for a range between 40 µm to 200 µm of each of the four dNTPs or a 1:2 ratio of dNTPs to magnesium

Impure water

Contamination may have occurred during previous pipetting events

• Use autoclaved and filtered nuclease-free ddH₂O.

Insufficient Mg²⁺

 Mg²⁺ acts as a cofactor to enhance DNA polymerase activity.

• Mg²⁺ concentration should be in the range of 1-3 mM; we recommend using 1.5mM Mg²⁺ in the final reaction
• Optimize Mg²⁺ concentration to maximize PCR yields by testing in 0.2-1 mM increments
• May need to increase Mg²⁺ concentration if high concentrations of EDTA, other metal chelators, or dNTP is present
• Thoroughly mix Mg²⁺ solution and buffer before adding to reaction mix
• Check the enzyme’s preference for Mg salt solutions – e.g. Pfu DNA polymerase works better with MgSO₄ than MgCl₂
• Mg²⁺ concentration should always be higher than dNTP concentration as dNTPs will bind with Mg²⁺, depleting the effective optimal concentration required for the reaction

Additives are needed

Some GC-rich DNA templates or primers have a higher risk of formation of secondary structures (primer-dimers, self-binding) that can inhibit the reaction.

• DMSO is useful for problematic amplifications as it disrupts secondary structure formation
• Do not exceed 10% of the total reaction volume

Excess PCR additives or co-solvents

Too much additive can start inhibiting enzyme activity or weaken the primer’s ability to bind the target DNA.

• Use the lowest additive concentration possible
• Adjust annealing temperature to improve primer binding (e.g. if DMSO is used, a lower annealing temperature is preferred as DMSO decreases the melting point of the primers)
• Add more DNA polymerase or use DNA polymerases with high processivity
• Use additives or co-solvents that are specifically formulated for the DNA polymerase you are using

dUTP or modified nucleotides in reaction mix

• Optimize the ratio of the modified nucleotides to dNTP to increase PCR efficiency
• Ensure that the DNA polymerase can incorporate the modified nucleotides

Missing reagents

•  Make sure all components are added – make a checklist
• Include a positive and negative control to verify that components are present and functional

Non-homogeneous reagents

•  Mix stocks thoroughly to avoid density gradients that can form during storage and setup
• Check that reagents are fully thawed

Contaminated reaction tubes or solutions

• Autoclave tubes to prevent contamination
• Prepare fresh solutions
• Use new reagents and tubes

Thermal cycler issues

• Check that the temperatures and times for your PCR program are correctly set
• Use a different cycler – calibration may be off
• Test calibration of heating block
• Ensure the thermocycler has a heated lid and it is turned on during the program

Too many cycles

• Increases the opportunity for non-specific amplification and errors
• Changes the pH of the reaction, destabilizing the DNA template
• Reduces the efficiency of the polymerase by increasing the amount of PCR product and errors
• Decreases the amounts of dNTPs causing an unbalanced concentration and increased chance of misincorporation

• Typically, 20-35 cycles are used
• If the template concentration is low, use more cycles; extend to 40 cycles if fewer than 10 copies of DNA are available
• If the template concentration is high, use fewer cycles

Denaturation temperature is too low or high

• Low temperature results in incomplete denaturation (double-stranded DNA may not denature)
• High temperatures may reduce enzyme activity

• Optimize the denaturation temperature
• Typically, a denaturation temperature of 94-98°C is used
• Use the temperature gradient function on your PCR machine to optimise the denaturation temperature in one PCR run. This function enables you to evaluate several different denaturation temperatures across a single block. 

Denaturation time is too long or too short

• Too long: the DNA may be degraded and the enzyme activity may be reduced
• Too short: the DNA will not be completely denatured

• Optimize the denaturation time
• For initial denaturation, use 3-5 min
• For denaturation during cycling, use 10-30s

• Typically, a 20-30 sec annealing time is recommended
• If non-specific products are shorter than target: increase annealing time
• If non-specific products are longer than target: decrease annealing time

• Typically, use an annealing temperature that is 3-5°C lower than the T_m of the primer
• Use a thermal gradient to optimize the annealing temperature stepwise in 1-2°C increments, starting at 5°C below the T_m (of the lower T_m if using pairs of primers)
• Use a touch-down PCR protocol

• Typically, a 30s-1 min/kb extension time is recommended
• Decrease extension time in small increments if the time is too long
• Increase extension time if target is long (>10 kb)
• Include a final extension step for 5-15 min

Complex template

E.g. Template contains GC-rich or secondary structures.

GC content that is  >65% is typically difficult to amplify due to its increased stability and resistance to denaturation.

• Increase the annealing temperature – optimize using a thermal gradient
• Use high processivity or hot-start DNA polymerases such as our MegaFi™ Pro Fidelity DNA Polymerase (Cat. No. G886) or BlasTaq™ HotStart DNA Polymerase (Cat. No. G896) – these PCR enzymes have a high affinity for DNA templates and are better for amplifying difficult targets
• Use a PCR additive or co-solvent, such as DMSO or secondary structure destabilizer – this denatures GC-rich sequences and secondary structures; do not exceed 10% per reaction sample
•  Increase denaturation time and/or temperature – this helps primer binding and enzyme thermostability; especially useful for GC-rich templates
• Use a touch-down PCR protocol

Incorrect template concentration

• For templates that are not complex (i.e. plasmid, lambda, BAC DNA): use 1 pg – 10 ng of DNA per 50 μL reaction
• For complex templates (i.e. genomic DNA): use 1 ng - 1 μg of DNA per 50 μL reaction
• Optimize the template amount using a dilution series
• Ensure that the template is thawed completely and well mixed

Target is too long

PCR component concentrations and/or cycling conditions may be insufficient for longer targets.

• Increase duration of PCR steps, especially the extension time
• Check the amplification length capability of the polymerase used
• Use polymerases designed for long PCR such as our MegaFi™ Pro Fidelity DNA Polymerase (Cat. No. G886) or BlasTaq™ HotStart DNA Polymerase (Cat. No.G896); choose DNA polymerases with high processivity
• Reduce the annealing and extension temperatures

Impure primers

Contaminants in primers may inhibit PCR.

• Use desalted primers or more highly purified primers
• Dilute primers to check for inhibitory effects

Concentrations of primers are too high

This increases the risk of primers binding non-specifically to the template or to each other

• Optimize primer concentration: Typically, a primer concentration of 0.1-1 μM is recommended.

Primers were designed or synthesized incorrectly

• High GC content in primers may result in primers binding to other sequences aside from the target or cause hairpins or primer dimers
• Primers that are too short or too long may also cause nonspecific binding

• Primers should be a minimum of 18 bp long and have a minimum melting temperature of 52℃
• Review primer design and verify that the primers have the correct sequence, are specific to the target of interest, and are complementary to the template
• Check that primers are non-complementary internally or to each other as this could cause formation of primer-dimers (self-primers)
• Use a primer design program or check out our primer design service
• Avoid GC-rich 3’ ends and ensure that primers have less than 60% GC content – aim for a maximum of 45% GC content
• Avoid stretches of 4 or more G nucleotides in a row as this may cause problems during primer synthesis
• Try to design the primers to end with a G or C nucleotide on the 3’ end, as this “GC clamp” can help create a stronger bond for the amplification to initiate
• Perform a BLAST search to avoid amplification of pseudogenes or unintended regions
• When working with genomic DNA, check for introns between primer sites as if the introns are too long, it may result in incomplete product/no amplification
• Use nested primers

Inappropriate DNA polymerase

If standard Taq is not sufficient for your template, there are many speciality enzymes available to try.

• Use a different polymerase: try our BlasTaq™ HotStart DNA Polymerase (Cat. No. G595). This PCR enzyme:
  • Prevents primer degradation that can occur due to the 3’- 5’ exonuclease activity of proofreading DNA polymerases
  • Eliminates nonspecific amplification, which increases yield
• Setup PCR on ice if using non-hot-start DNA polymerases and add polymerase last to reaction mix to ensure amount is completely transferred and premature activation does not occur

Excess DNA polymerase

• Review recommended amounts as provided by the manufacturer and decrease accordingly.

Impure dNTPs

• Use high-quality dNTPs such as our dNTP mix.

Impure water

• Use autoclaved and filtered nuclease-free ddH₂O.

Excess Mg²⁺

Excess magnesium stabilizes primer binding too much and can result in more nonspecific primer binding

• Reduce the Mg²⁺ concentration in the final reaction (while ensuring it remains higher than the dNTP concentration)
• Mg²⁺ concentration should be approximately 1-3 mM
• Optimize concentration by adjusting the amount in 0.2-1 mM increments

Premature replication

• Use a hot-start polymerase such as our BlasTaq™ HotStart DNA Polymerase (Cat. No. G595) which contains a proprietary antibody that blocks polymerase activity at low temperatures
• Set up reactions on ice and use chilled components
• Preheat thermocycler to the denaturation temperature before adding samples

Ramping speed of thermal cycler is too slow

This lowers the reaction temperature, leaving  time for non-specific binding to occur.

• Increase ramping speed to the maximum rate for the machine.

Thermal cycler issues

• Check that the temperatures and times for your PCR program are correctly set
• Use a different cycler – calibration may be off
• Test calibration of heating block
• Ensure the thermocycler has a heated lid and it is turned on during the program

DNA contamination of stocks

Non-specific bands may be from foreign DNA.

• Add template to the reaction setup last to minimize exposure of your sample to environmental contaminants
•  Include a positive and negative (no template) control to verify source of the contamination
• Use positive displacement pipettes or non-aerosol tips
• Have a dedicated workspace and pipettes for PCR setup
• Use new stocks
• Use autoclaved PCR vials
• Wear clean gloves and lab coat

Contamination

Negative samples are more prone to contamination.

• Work in a clean and sterile and work area
• Use filtered pipette tips
• Change tips frequently to help avoid any cross-contamination
• Always prepare negative control tube first and close the lid of the tube completely before preparing the positive control tubes

Too many cycles

• Intense background smearing with indistinguishable bands is an indicator of over cycling which increases risk of nonspecific amplification/errors
• A blank negative control indicates that the smearing is caused by suboptimal PCR conditions (optimization is needed)

• Typically, 20-35 cycles are used
• If the template concentration is low, use more cycles; extend to 40 cycles if fewer than 10 copies of DNA are available
• If the template concentration is high, use fewer cycles

Denaturation temperature is too low or high

• Low temperature results in incomplete denaturation (double-stranded DNA may not denature)
  High temperatures may reduce enzyme activity

• Optimize the denaturation temperature
• Typically, a denaturation temperature of 94-98oC is used
• Use the temperature gradient function on your PCR machine to optimise the denaturation temperature in one PCR run. This function enables you to evaluate several different denaturation temperatures across a single block. 

Denaturation time is too long or too short

• Too long: the DNA may be degraded and the enzyme activity may be reduced
• Too short: the DNA will not be completely denatured

• Optimize the denaturation time
• For initial denaturation, use 3-5 min
• For denaturation during cycling, use 10-30s

Annealing time is too short

• Typically, an annealing time of 20- 30 sec is recommended

Annealing temperature is too low

• Typically, use an annealing temperature that is 3-5oC lower than the Tm of the primers
• Use a thermal gradient to optimize the annealing temperature stepwise in 1-2oC increments, starting at 5oC below the Tm (of the lower Tm, if using pairs of primers)
• Adjust the annealing temperature if you are using a PCR additive or co-solvent
• Use a touch-down PCR protocol

Extension time is too short

There is insufficient time for complete replication of the target.

• Typical extension times are 30s-1 min/kb
• Longer templates will need long extension times, or consider  using a faster enzyme such as our BlastTaq™ DNA Polymerase (Cat. No. G894) which is 3X faster than regular Taq
• Include a final extension time of at least 5 min

Extension time is too long

Overly long extension times can inactivate the PCR enzyme.

• Reduce the temperature by 3-4°C – this will help with the thermostability of the polymerase and keep it active, especially when amplifying longer targets (>10 kb)

Poor template quality or contamination from an exonuclease

The template could have been degraded or sheared and may appear as smears or lead to intense background.

• If possible, use fresh template or re-make the template DNA (especially if it is genomic DNA as old stocks are prone to degradation or shearing)
• Minimize shearing and nicking of gDNA during isolation by minimizing vortexing and freeze-thawing
• Store DNA in TE buffer (pH 8.0) to prevent degradation by nucleases
• Before and after incubation with Mg²⁺, analyze DNA via gel electrophoresis to evaluate the integrity of the template, if necessary
• Limit or avoid UV exposure of DNA
• Use dyes with a longer-wavelength excitation to visualize DNA; irradiation from longer-wavelengths are less damaging to the DNA

Excess template

• May bind all the primers in early amplification cycles and result in insufficient primers for subsequent rounds of amplification
• Polymerase can be inhibited from inhibitor compounds carried over from the template
• Can cause inefficient denaturation

• Use different concentrations of the template via a dilution series to optimize the input amount; typically a range of 100-200 ng of mammalian genomic DNA is used
• Increase the denaturation time and temperature
• Increase annealing temperature

Target is too long

PCR component concentrations and/or cycling conditions may be insufficient for longer targets.

• Increase duration of PCR steps, especially the extension time
• Check the amplification length capability of the polymerase used
• Use polymerases designed for long PCR such as our MegaFi™ Pro Fidelity DNA Polymerase (Cat. No. G886) or BlasTaq™ HotStart DNA Polymerase (Cat. No.G896); choose DNA polymerases with high processivity
• Reduce the annealing and extension temperatures

Impure primers

Contaminants in primers may inhibit PCR.

• Use desalted primers or more highly purified primers
• Dilute primers to check for inhibitory effects

Incorrect primer design/synthesis

Poor primer design will result in inefficient amplification of the DNA template.

• Primers should be a minimum of 18 bp long and have a minimum melting temperature of 52℃
• Review primer design and verify that the primers have the correct sequence, are specific to the target of interest, and are complementary to the template
• Check that primers are non-complementary internally or to each other as this could cause formation of primer-dimers (self-primers)
• Use a primer design program or check out our primer design service
• Avoid GC-rich 3’ ends and ensure that primers have less than 60% GC content – aim for a maximum of 45% GC content
• Avoid stretches of 4 or more G nucleotides in a row as this may cause problems during primer synthesis
• Try to design the primers to end with a G or C nucleotide on the 3’ end, as this “GC clamp” can help create a stronger bond for the amplification to initiate
• Perform a BLAST search to avoid amplification of pseudogenes or unintended regions
• When working with genomic DNA, check for introns between primer sites as if the introns are too long, it may result in incomplete product/no amplification
• Use nested primers

Inappropriate DNA polymerase

If standard Taq is not sufficient for your template, there are many speciality enzymes available to try.

• Use a different polymerase: try our BlasTaq™ HotStart DNA Polymerase (Cat. No. G595). This PCR enzyme:
  • Prevents primer degradation that can occur due to the 3’- 5’ exonuclease activity of proofreading DNA polymerases
  • Eliminates nonspecific amplification, which increases yield
• Setup PCR on ice if using non-hot-start DNA polymerases and add polymerase last to reaction mix to ensure amount is completely transferred and premature activation does not occur

Excess Mg²⁺

Excess magnesium stabilizes primer binding too much and can result in more nonspecific primer binding.

• Reduce the Mg²⁺ concentration in the final reaction (while ensuring it remains higher than the dNTP concentration)
• Mg²⁺ concentration should be approximately 1-3 mM
• Optimize concentration by adjusting the amount in 0.2-1 mM increments

Impure dNTPs

• Use high-quality dNTPs such as our dNTP mix.

Impure water

• Use autoclaved and filtered nuclease-free ddH₂O.

Contamination

• Aliquot each component to reduce risk of contamination
• Work on a clean bench, use filtered pipette tips, and change tips frequently, wear clean gloves and lab coat
• Include a positive and negative (no template) control to verify contamination source

Ramping speed of the thermal cycler is too slow

• Increase ramping speed to the maximum rate for the machine.

Issue with agarose gel electrophoresis

• Cool down agarose solution before adding ethidium bromide or DNA-binding dye and thoroughly mix before pouring into the gel cassette to ensure formation of good quality gel.

Too much product loaded on gel

• Load less of the product.

Incorrect annealing temperature

• Typically, use an annealing temperature that is 3-5°C lower than the T_m of the primer
• Use a thermal gradient to optimize the annealing temperature stepwise in 1-2°C increments, starting at 5°C below the T_m (of the lower T_m if using pairs of primers)
• Use a touch-down PCR protocol

Incorrect primer design/synthesis

Poor primer design will result in inefficient amplification of the DNA template.

• Primers should be a minimum of 18 bp long and have a minimum melting temperature of 52℃
• Review primer design and verify that the primers have the correct sequence, are specific to the target of interest, and are complementary to the template
• Check that primers are non-complementary internally or to each other as this could cause formation of primer-dimers (self-primers)
• Use a primer design program or check out our primer design service
• Avoid GC-rich 3’ ends and ensure that primers have less than 60% GC content – aim for a maximum of 45% GC content
• Avoid stretches of 4 or more G nucleotides in a row as this may cause problems during primer synthesis
• Try to design the primers to end with a G or C nucleotide on the 3’ end, as this “GC clamp” can help create a stronger bond for the amplification to initiate
• Perform a BLAST search to avoid amplification of pseudogenes or unintended regions
• When working with genomic DNA, check for introns between primer sites as if the introns are too long, it may result in incomplete product/no amplification
• Use nested primers

Incorrect Mg²⁺ concentration

• Should be approximately 1-3 mM
• Adjust the amount in 0.2-1 mM increments
• Concentration should be always higher than dNTP concentration

Nuclease contamination

• Repeat PCR using fresh reagents.

Concentration of primers is too high

This may increase risk of primers binding to each other.

• Typically, a primer concentration of 0.2-1 μM is recommended
• Re-design primers to ensure minimal self and complementary binding (refer to our article on best practices for primer design)

Poor primer design or incorrect synthesis 

Complementary sequences or consecutive G or C nucleotides at the 3’ ends of primers may cause primer-dimers to form.

• Primers should be a minimum of 18 bp long and have a minimum melting temperature of 52℃
• Review primer design and verify that the primers have the correct sequence, are specific to the target of interest, and are complementary to the template
• Check that primers are non-complementary internally or to each other as this could cause formation of primer-dimers (self-primers)
• Use a primer design program or check out our primer design service
• Avoid GC-rich 3’ ends and ensure that primers have less than 60% GC content – aim for a maximum of 45% GC content
• Avoid stretches of 4 or more G nucleotides in a row as this may cause problems during primer synthesis
• Try to design the primers to end with a G or C nucleotide on the 3’ end, as this “GC clamp” can help create a stronger bond for the amplification to initiate
• Perform a BLAST search to avoid amplification of pseudogenes or unintended regions
• When working with genomic DNA, check for introns between primer sites as if the introns are too long, it may result in incomplete product/no amplification
• Use nested primers

Too many cycles

• Reduces polymerase efficiency due to increasing the amount of PCR product and errors
• Decreases the amounts of dNTPs causing an unbalanced concentration and increased chance of misincorporation

• Do not exceed 30 cycles
• Increase amount of input DNA or decrease number of cycles

Low fidelity DNA polymerase was used

• Instead of using standard Taq, use a high fidelity polymerase such as our MegaFi™ Pro Fidelity DNA Polymerase (Cat. No. G886) which offers 1,300X less error rates.

Extension time is too long

• Typically, an extension time of 30s-1 min/kb is recommended
• Decrease extension time in small increments and test

Damaged or degraded template

The template may have been sheared or degraded.

• If possible, use fresh template or re-make the template DNA (especially if it is genomic DNA as old stocks are prone to degradation or shearing)
• Minimize shearing and nicking of gDNA during isolation by minimizing vortexing and freeze-thawing
• Store DNA in TE buffer (pH 8.0) to prevent degradation by nucleases
• Before and after incubation with Mg²⁺, analyze DNA via gel electrophoresis to evaluate the integrity of the template, if necessary
• Limit or avoid UV exposure of DNA
• Use dyes with a longer-wavelength excitation to visualize DNA; irradiation from longer-wavelengths are less damaging to the DNA

Incorrect primer design/synthesis

If there are direct repeats within primer sequences, multiple repeats at the ends of PCR products may appear due to sequence misalignment.

• Primers should be a minimum of 18 bp long and have a minimum melting temperature of 52℃
• Review primer design and verify that the primers have the correct sequence, are specific to the target of interest, and are complementary to the template
• Check that primers are non-complementary internally or to each other as this could cause formation of primer-dimers (self-primers)
• Use a primer design program or check out our primer design service
• Avoid GC-rich 3’ ends and ensure that primers have less than 60% GC content – aim for a maximum of 45% GC content
• Avoid stretches of 4 or more G nucleotides in a row as this may cause problems during primer synthesis
• Try to design the primers to end with a G or C nucleotide on the 3’ end, as this “GC clamp” can help create a stronger bond for the amplification to initiate
• Perform a BLAST search to avoid amplification of pseudogenes or unintended regions
• When working with genomic DNA, check for introns between primer sites as if the introns are too long, it may result in incomplete product/no amplification
• Use nested primers

Poor quality of primers

• Remove DNA oligos that are not full length.

Excess Mg²⁺

May impact the proofreading activity of enzymes and increases chances of misincorporation of dNTPs.

• Reduce the Mg²⁺ concentration in the final reaction (while ensuring it remains higher than the dNTP concentration)
• Mg²⁺ concentration should be approximately 1-3 mM
• Optimize concentration by adjusting the amount in 0.2-1 mM increments

Unbalanced concentration of dNTPs

Increases the PCR error rate due to higher risk of dNTP misincorporation.

• Prepare fresh dNTP mixes
• Fully thaw and thoroughly mix before adding dNTPs to reaction mix
• Ensure the concentrations of dNTPs are equal
• Use a readymade dNTP mix

Desired template sequence may be toxic to host

• Clone into a non-expression (promoter-less) vector with a low-copy number to reduce sequence errors in your template caused by the toxic burden on the host.

Pipette issue

• Recalibrate your pipette and ensure good pipetting technique and accurate volume transfer.

Machine settings are incorrect

• Check whether ROX is required for your machine (by consulting the thermocycler manual)  and add the correct amount needed
• Ensure that you are selecting the appropriate probe settings on your machine (e.g. SYBR vs. Taqman Probe settings)
• Ensure you have entered the settings into your machine correctly (e.g. Users may sometimes accidentally set target reporter to “ROX” and passive reference dye to “None” when they should have set target reporter to “SYBR” and passive reference dye to “ROX”.

Incorrect primer design/synthesis

If there are direct repeats within primer sequences, multiple repeats at the ends of PCR products may appear due to sequence misalignment

• Primers should be a minimum of 18 bp long and have a minimum melting temperature of 52℃
• Review primer design and verify that the primers have the correct sequence, are specific to the target of interest, and are complementary to the template
• Check that primers are non-complementary internally or to each other as this could cause formation of primer-dimers (self-primers)
• Use a primer design program or check out our primer design service
• Avoid GC-rich 3’ ends and ensure that primers have less than 60% GC content – aim for a maximum of 45% GC content
• Avoid stretches of 4 or more G nucleotides in a row as this may cause problems during primer synthesis
• Try to design the primers to end with a G or C nucleotide on the 3’ end, as this “GC clamp” can help create a stronger bond for the amplification to initiate
• Perform a BLAST search to avoid amplification of pseudogenes or unintended regions
• When working with genomic DNA, check for introns between primer sites as if the introns are too long, it may result in incomplete product/no amplification
• Use nested primers

Carryover or crossover contamination

• Use filtered pipette tips with aerosol barriers
• Dedicate separate work area for PCR set up and decontaminate after each use
• Leave pipettes and other equipment under UV light – UV irradiation damages residual DNA by promoting the cross-linking of thymidine residues
• Spray work area and equipment with 10% bleach and wipe clean

Compare Performance:

Product Features:

• Enable streamlined protocol in a simple reaction set-up
• Allow accurate quantification of a variety of gene targets
• Reduce pipetting steps to minimize the risk of contamination
• Contains dye comparable to SYBR™ Green and EvaGreen™
• Free Samples available

Applications:

• Gene-expression analysis
• Population genotyping

Compare Performance:

Product Features:

• Designed for TaqMan Probe-based qPCR
• 40-70% less time than regular Taq
• 6 kb/min extension speed
• Highly robust and sensitive
• Free Samples available

Applications:

• Gene-expression analysis
• Population genotyping

Compare Performance:

Product Features:

• Simple, one-step RT-qPCR reaction
• Flexibility with RNA templates and primer selection
• ROX reference dye provided separately
• Universally compatible with most qPCR instruments
• Free Samples available

Applications:

• Gene-expression analysis
• Population genotyping

Compare Performance:

MegaFi™ Pro Fidelity DNA Polymerase (Cat. No. G886) has ~1300X better proofreading than other enzymes on the market.

A lacZa-containing sequence was amplified using various DNA polymerases, subcloned, and plated on X-gal-containing media. Colonies without PCR amplified lacZa mutations appear dark blue, while those that have PCR introduced frameshift mutations appear white. Blue vs. white colonies were counted and relative fidelity was calculated relative to Taq DNA Polymerase.

Product Features:

• 1300X lower error rates compared to regular Taq
• 3X more cost-effective at only $0.48 USD/reaction
• Amplifies difficult templates
• Effective in long-range PCR
• Free Samples available

Applications:

• Ultra high-fidelity PCR
• Next Generation Sequencing

Compare Performance:

MegaFi™ Fidelity DNA Polymerase (Cat. No. G896) has ~1300X better proofreading than other enzymes on the market.

A lacZa-containing sequence was amplified using various DNA polymerases, subcloned, and plated on X-gal-containing media. Colonies without PCR amplified lacZa mutations appear dark blue, while those that have PCR introduced frameshift mutations appear white. Blue vs. white colonies were counted and relative fidelity was calculated relative to Taq DNA Polymerase.

Product Features:

• 1300X lower error rates compared to regular Taq
• 3X more cost-effective at only $0.48 USD/reaction
• Amplifies difficult templates
• Effective in long-range PCR
• Free Samples available

Applications:

• Ultra high-fidelity PCR
• Next Generation Sequencing

Compare Performance:

BlasTaq™ DNA Polymerase enables extension speeds as fast as 6 kb/min.

Total reaction times for abm’s BlasTaq™ DNA Polymerase (Cat. No. G894) and Taq were determined for the amplification of different sized amplicons: 500 bp, 1 kb, 2 kb, and 5 kb. Reaction times are based on a 30-cycle program using the recommended reaction protocol for each enzyme.

Product Features:

• 6 kb/min extension speeds
• 70% less time needed than regular Taq
• Superior sensitivity
• Can be used with TA cloning vectors
• Free Samples available

Applications:

• Routine PCR
• TA cloning

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BlasTaq™ DNA Polymerase enables extension speeds as fast as 6 kb/min.

Total reaction times for abm’s BlasTaq™ DNA Polymerase (Cat. No. G894) and Taq were determined for the amplification of different sized amplicons: 500 bp, 1 kb, 2 kb, and 5 kb. Reaction times are based on a 30-cycle program using the recommended reaction protocol for each enzyme.

Product Features:

• 6 kb/min extension speeds
• 70% less time needed than regular Taq
• Superior sensitivity
• Can be used with TA cloning vectors

Applications:

• Routine PCR
• TA cloning

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BlasTaq™ HotStart DNA Polymerase eliminates non-specific amplification.

abm’s BlasTaq™ HotStart DNA Polymerase (Cat. No. G595) was used to amplify a 7 kb target. The antibody concentration was decreased incrementally from lanes 1-5. Lane 1 is our BlasTaq™ HotStart formulation and lane 5 contains no antibody.

Product Features:

• Contains proprietary antibody that blocks polymerase activity at low temperatures
• 70% less time needed than regular Taq
• No non-specific amplification or primer-dimers
• Improves yield of desired product
• Streamlined, simple reaction set-up
• Free Samples available

Applications:

• Difficult templates
• TA cloning

Compare Performance:

Product Features:

• Contains proprietary antibody that blocks polymerase activity at low temperatures
• 70% less time needed than regular Taq
• No non-specific amplification or primer-dimers
• Improves yield of desired product
• Streamlined, simple reaction set-up
• Free Samples available

Applications:

• Difficult templates
• TA cloning

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PCR amplification using abm’s Taq DNA Polymerase (Cat. No. G009) of various target sizes ranging from 560 bp to 6500 bp followed by electrophoresis on a 1% agarose gel.

Product Features:

• Consistent results over a wide range of DNA templates
• Excellent yield and sensitivity
• Affordable choice, at only $0.20 USD/ reaction

Applications:

• Routine PCR

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PCR amplification using abm’s Taq DNA Polymerase (Cat. No. G009) of various target sizes ranging from 560 bp to 6500 bp followed by electrophoresis on a 1% agarose gel.

Product Features:

• Consistent results over a wide range of DNA templates
• Excellent yield and sensitivity
• Contains gel loading dye for direct loading of PCR products onto agarose dye
• Affordable choice, at only $0.25 USD/ reaction

Applications:

• Routine PCR

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OneScript® Plus Reverse Transcriptase can elongate RNA templates up to 15kb in length. OneScript® Plus Reverse Transcriptase (Cat. No. G237) was used in a reaction with a range of human RNA fragments. The resulting synthesized cDNA was followed by PCR and visualized on a 1% agarose gel. 

Product Features:

• High thermostability at 50-55°C/122-131°F
• Synthesize 15 kb RNA Templates
• 10-15 minutes reaction time
• OneScript® Plus cDNA Synthesis Kit (Cat. No. G236) also available
• Pair with BlasTaq™ 2X PCR MasterMix (Cat. No. G895) for Two-Step RT-PCR workflows

Applications:

• Synthesizing cDNA from a ssRNA
• DNA primer extension
• Sequencing dsDNA
• Constructing cDNA library
• Producing template for use in RT-PCR or real-time RT-PCR
• Generating probes for hybridization

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OneScript® Hot Reverse Transcriptase has exceptional thermostability. OneScript® Hot Reverse Transcriptase (Cat. No. G593) was used in a 10 min. reaction with RNA template at temperatures ranging from 50 and 80 °C. The resulting synthesized cDNA was followed by PCR and visualized on a 1% agarose gel.

Product Features:

• Thermostable from 60-72°C/140-161°F
• Transcribes degraded samples and resists inhibitors/contaminants
• Provides high cDNA yields from difficult or ultra-low RNA samples

Applications:

• RT-PCR

Compare Performance:

OneScript® Plus Reverse Transcriptase can elongate RNA templates up to 15kb in length. OneScript® Plus Reverse Transcriptase (Cat. No. G237) was used in a reaction with a range of human RNA fragments. The resulting synthesized cDNA was followed by PCR and visualized on a 1% agarose gel. 

Product Features:

• Maximal flexibility in priming – oligo(dT), random primers or gene-specific primers
• Robust cDNA synthesis from any RNA template
• High reproducibility and excellent yield
• Employs abm's OneScript® Plus Reverse Transcriptase (Cat. No. G237)

Applications:

• Synthesizing cDNA from a ssRNA
• DNA primer extension
• Sequencing dsDNA
• Constructing cDNA library
• Producing template for use in RT-PCR or real-time RT-PCR
• Generating probes for hybridization

Compare Performance:

OneScript® Hot Reverse Transcriptase (Cat. No. G593) retains exceptional activity even after 7 days at 37ºC. OneScript® Hot Reverse Transcriptase was maintained at either 1 day or 7 days and at either 4ºC, 25ºC, or 37ºC. The enzyme was then used in a reaction to amplify two 1kb RNA templates (target A and target B). The resulting synthesized cDNA was followed by PCR and visualized on a 1% agarose gel.

Product Features:

• Extremely thermostable cDNA synthesis at 60-72ºC
• Transcribes degraded samples and resists inhibitors/contaminants
• High cDNA yields from difficult or ultra-low RNA samples

Applications:

• RT-PCR

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All-in-One 5X RT MasterMix (Cat. No. G592) ensures accurate qPCR results where contaminating gDNA would otherwise result in artificially-early Ct values. RNA samples contaminated with gDNA were reverse-transcribed for 25 min with All-in-One 5X RT MasterMix. 1µl of RT product was then used in qPCR to assay amplification of a GAPDH target.

Product Features:

• Easy one-step setup reduces pipetting and sample handling
  High cDNA yields from difficult samples with OneScript® Hot Reverse Transcriptase (Cat. No. G593)
• Inhibits ribonuclease contaminants with RNaseOFF Ribonuclease Inhibitor (Cat. No. G591)
• Removes contaminating gDNA with temperature-sensitive DNase
• Also includes dNTPs, Oligo (dT)s, and random primers

Applications:

• COVID-19 research
• RT-PCR and RT-qPCR
• in vitro transcription
• cDNA synthesis
• Long-term RNA storage

Product Features:

• Easy one-step setup reduces pipetting and sample handling
  High cDNA yields from difficult samples with OneScript® Hot Reverse Transcriptase (Cat. No. G593)
• Inhibits ribonuclease contaminants with RNaseOFF Ribonuclease Inhibitor (Cat. No. G591)
• Also includes dNTPs, Oligo (dT)s, and random primers

Applications:

• COVID-19 research
• RT-PCR and RT-qPCR
• in vitro transcription
• cDNA synthesis
• Long-term RNA storage

Product Features:

• Enjoy simple, one-step RT-PCR reactions
• 1300X lower error rates due to our MegaFi™ Pro Fidelity DNA Polymerase (Cat. No. G896)
• Extremely thermostable cDNA synthesis at 50-80°C due to our OneScript® Hot Reverse Transcriptase (Cat. No. G593)
• Excellent protection against common ribonucleases due to our RNaseOFF Ribonuclease Inhibitor (Cat. No. G591)

Applications:

• RT-PCR