| Human error |
- Confirm all reagents were added appropriately at their respective steps.
- Ensure no reagents were contaminated or expired.
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| PCR reagents and additives |
- Prepare new reagents (e.g., fresh working stocks, new dilutions), and then systematically add one new reagent at a time to reaction mixtures to determine the culprit.
- Very old DNA can often accumulate inhibitors so the addition of bovine serum albumin may help alleviate the problem.
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| Primer dimer formation or hairpin loop structures form with the primers |
- Will limit the amount of product produced.
- Can be confirmed by gel electrophoresis, and are visible as small band on the gel of < 100 b near the bottom of the lanes.
- Alter the ratio of template to primer, and decrease the primer concentration if it is in severe excess to the template concentration.
- Add DMSO to the reaction.
- Use a hot start thermal cycling method instead.
- Design new primers.
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| Nonspecific products are produced |
- Nonspecific products can be confirmed by a ladder effect on the agarose gel and are bands that migrate at a different size than the desired product.
- A smear on the gel may also indicate that primers are annealing to multiple spots in the DNA outside of the target amplicon.
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| Concentration of Mg2+ must be adjusted | - Generally, the PCR product yield will increase with the addition of greater concentrations of Mg2+, though this will also decrease the specificity and fidelity of the DNA polymerase.
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| Concentration of KCl must be adjusted |
- Longer PCR products (> 10 kb) benefit from reducing KCl from its normal 50 mM reaction concentration, often with the addition of DMSO and/or glycerol.
- If the amplicon is < 1000 bp and long non-specific products are forming, specificity may be improved by titrating KCl, increasing the concentration in 10 mM increments up to 100 mM.
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| Concentration of Deoxynucleotide 5'-triphosphates (dNTPs) is inhibiting PCR |
- Ensure that dNTP concentration, for each G, C, A and T is between 20-200 µM.
- Lower concentrations of dNTPs may increase specificity and fidelity of the reaction.
- For longer PCR-fragments, a higher dNTP concentration may be required.
- A large change in the dNTP concentration may necessitate a change in the concentration of Mg2+.
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| GC content is too high |
- Analyze the GC content in the amplicon.
- GC content >60% may inhibit the reaction, and may require an additive including DMSO, formamide, and dc7GTP.
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