Nonspecific amplification, no amplification, wrong product size
Curious result? Consult our PCR Troubleshooting Guide after your reaction to identify potential causes of unexpected results and solutions. More details on reaction conditions and setup optimization can be found in our Guidelines for PCR Optimization with Thermophilic DNA Polymerases and this blog post.
Technical Support is always happy to work with you to troubleshoot your PCR. If you would like assistance, you can:
- Email us at info@neb.com
- Call Technical Support at (800)-0632-7799, available Monday through Friday, 9:00AM - 6:00PM EST
- Fill out this webform
Failure to amplify a target greater than 5 kb
If you are struggling to amplify a target that is greater than 5 kb, try some of these tips:
- We recommend using Q5®, Phusion®, or LongAmp® polymerases
- If using Q5, try decreasing the final primer concentration to 150-300nM
- Stand-alone enzyme + buffer formulations allow more flexibility in reaction optimization than master mixes
- Use more template
- Treat the purified template gently as not to shear it
- Optimize enzyme concentration by testing a titration of enzyme in the reaction (0.25-2 units/50μl reactions)
- Increase the number of cycles
- Lengthen extension time to 40s/kb
Smearing on an agarose gel
When PCR conditions are not optimal, a smear or high level of background is often observed. Try one or more of the following suggestions:
- Use less enzyme
- Decrease the extension temperature to 3°C below the extension temperature recommended by the specific product protocol
- For example, the OneTaq® protocol recommends a 68°C extension temperature; try 65°C.
- Raise the annealing temperature
- Try 2-step cycling protocols
If there is an illuminated halo around the well in addition to smearing from the well, use less template.
