If no amplification products are obtained, what parameters should be considered first when troubleshooting?
If no amplification products are obtained, what parameters should be considered first when troubleshooting?
1. Consider the following:
-First, ensure that all PCR components were included in the reactions. A positive control should always be included to ensure that each component is present and functional.
-If there were no problems with the experimental setup, increase the number of PCR cycles (3–5 cycles at a time), up to 40 cycles. Increasing the cycle number can overcome issues with a low-abundance template or template inaccessibility due to impurities in or poor priming efficiency of the primers.
-If increasing the cycle number does not improve results, the PCR conditions might be too stringent for the particular primer set or template. Consider modifying the PCR conditions as follows:
*Lower the annealing temperature in increments of 2 degrees.
*Increase the extension time.
*Increase the template amount. Refer to the guidelines provided with the enzyme to determine the optimal amount of template.
2. Consider these additional possible reasons for PCR failure:
-PCR inhibitors in the template sample. If PCR inhibitors are present, using diluted template may increase PCR efficiency. Alternatively, the template may need to be purified using a kit such as the NucleoSpin Gel and PCR Clean-up kit. If purifying the template is not a possibility, an enzyme that has a higher tolerance to impurities, such as Terra PCR Direct polymerase, may improve results.
-The template has >65% GC content. When amplifying from templates with high GC content, use an enzyme formulated for this condition. Visit our PCR selection guide to find an appropriate enzyme.
-Primers are not optimal. Check your primers carefully; redesign if necessary. Also, consider re-amplifying the primary PCR product using 10-fold dilutions (1:100 to 1:10,000) using nested primers.
3. When using PrimeSTAR HS DNA Polymerase, consider:
-Using an appropriate amount of template. If the template is human genomic DNA or a cDNA library, use no more than ~100 ng of the template in a 50-µl reaction mixture.
-Using an extension time of at least 1 min/kb.
-Increasing the concentration of the primers.
4. When using PrimeSTAR Max DNA Polymerase, consider:
-Adjusting the extension time if the reaction mixture contains excess template. If the amount of template exceeds 200 ng in a 50-µl reaction mixture, set the extension time between 30 sec/kb and 1 min/kb.
-Increasing the concentration of the primers.
5. When using SpeedSTAR HS DNA Polymerase, consider:
-Increasing the extension time. Although the standard extension time is 10 sec/kb, the extension time can be increased to ~0.5 min/kb for complex templates such as human genomic DNA.