Sequence Errors
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Low fidelity polymerase |
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| Suboptimal reaction conditions |
- Reduce number of cycles
- Decrease extension time
- Decrease Mg++ concentration in the reaction
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| Unbalanced nucleotide concentrations |
- Prepare fresh deoxynucleotide mixes
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| Template DNA has been damaged |
- Start with a fresh template
- Try repairing DNA template with the PreCR® Repair Mix (NEB #M0309)
- Limit UV exposure time when analyzing or excising PCR product from the gel
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| Desired sequence may be toxic to host |
- Clone into a non-expression vector
- Use a low-copy number cloning vector
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Incorrect Product Size
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Incorrect annealing temperature |
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| Mispriming |
- Verify that primers have no additional complementary regions within the template DNA
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| Improper Mg++ concentration |
- Adjust Mg++ concentration in 0.2–1 mM increments
|
| Nuclease contamination |
- Repeat reactions using fresh solutions
|
No Product
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Incorrect annealing temperature |
- Recalculate primer Tm values using the NEB Tm calculator
- Test an annealing temperature gradient, starting at 5°C below the lower Tm of the primer pair
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| Poor primer design |
- Check specific product literature for recommended primer design
- Verify that primers are non-complementary, both internally and to each other
- Increase length of primer
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| Poor primer specificity |
- Verify that oligos are complementary to proper target sequence
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| Insufficient primer concentration |
- Primer concentration can range from 0.05–1 µM in the reaction. Please see specific product literature for ideal conditions
|
| Missing reaction component |
|
| Suboptimal reaction conditions |
- Optimize Mg++ concentration by testing 0.2–1 mM increments
- Thoroughly mix Mg++ solution and buffer prior to adding to the reaction
- Optimize annealing temperature by testing an annealing temperature gradient, starting at 5°C below the lower Tm of the primer pair
|
| Poor template quality |
- Analyze DNA via gel electrophoresis before and after incubation with Mg++
- Check 260/280 ratio of DNA template
|
| Presence of inhibitor in reaction |
- Further purify starting template by alcohol precipitation, drop dialysis or commercial clean up kit
- Decrease sample volume
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| Insufficient number of cycles |
- Rerun the reaction with more cycles
|
| Incorrect thermocycler programming |
- Check program, verify times and temperatures
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| Inconsistent block temperature |
- Test calibration of heating block
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Contamination of reaction tubes
or solutions |
- Autoclave empty reaction tubes prior to use to eliminate biological inhibitors
- Prepare fresh solutions or use new reagents and new tubes
|
| Complex template |
- Use Q5 High-Fidelity (NEB #M0491) or OneTaq® DNA Polymerases (NEB #M0480)
- For GC-rich templates, use Q5 High-Fidelity (NEB #M0491) or OneTaq® DNA Polymerases. Include the appropriate GC enhancer.
- For longer templates, we recommend LongAmp® Taq DNA Polymerase or Q5 high-Fidelity DNA polymerase or Q5 Hot-Start High-Fidelity DNA Polymerase (NEB #M0493)
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Multiple or Non-Specific Products
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Premature replication
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- Use a hot start polymerase, such as OneTaq Hot Start DNA Polymerase
- Set up reactions on ice using chilled components and add samples to thermocycler preheated to the denaturation temperature
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| Primer annealing temperature too low |
- Increase annealing temperature
|
| Incorrect Mg++ concentration |
- Adjust Mg++ in 0.2–1 mM increments
|
| Poor primer design |
- Check specific product literature for recommended primer design
- Verify that primers are non-complementary, both internally and to each other
- Increase length of primer
- Avoid GC-rich 3´ ends
|
| Excess primer |
- Primer concentration can range from 0.05–1 µM in the reaction. Please see specific product literature for ideal conditions.
|
| Contamination with exogenous DNA |
- Use positive displacement pipettes or non-aerosol tips
- Set-up dedicated work area and pipettor for reaction setup
- Wear gloves during reaction setup
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| Incorrect template concentration |
- For low complexity templates (i.e. plasmid, lambda, BAC DNA), use 1 pg–10 ng of DNA per 50 µl reaction
- For higher complexity templates (i.e. genomic DNA), use 1 ng–1 µg of DNA per 50 µl reaction
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