What could make a PCR product not amplify?

Introduction

If you've ever tried doing a PCR experiment and things just didn't work out, then this post is for you. I'll explain why the PCR product might not be amplifying, what causes it and how to troubleshoot your current experiments so that they can be successful!

The thermal cycler may be malfunctioning. Check the block temperature of the thermal cycler.

The thermal cycler may be malfunctioning. Check the block temperature of the thermal cycler by looking at the thermocouple reading on your digital display. If it is not within 0-5°C or 2-10°F of 95°C (or 50°C), check to see if:

• The temperature setting is correct
• The thermocouple is working properly
• You have plugged in your device, turned it on and allowed it to heat up before beginning your experiment

Contamination with product from previous reaction. Re-prepare primers, dNTPs and buffers.

In this case, the first step is to re-prepare the primers, dNTPs and buffers. Make sure there is no contamination from previous reactions. Also make sure that you use new primers. If you are using old ones, they may have degraded over time and are now inactive. You can also use new template DNA as well (new bacterial culture or PCR product).

The primers are wrong and simply don't match the template. Use a different set of primers.

If your primers are wrong, they may be designed to match a region that is not present in the template. This can happen if you have accidentally used a different sample as your template or if there was some mistake in ordering or making your primers.

When designing primers, make sure you are using at least 20 bases of sequence from each end of the region of interest to help avoid this issue. Make sure the primer's sequence matches the template sequence exactly and amplifies only that region!

The problem may be that the primers are too far apart to amplify a PCR product. Try redesigning one of your primer sets so that it is closer together and repeat your experiment.

PCR primers are designed to anneal to their target sequence and amplify DNA. If you have the right primer pair, but a PCR product is not amplified, it could be due to any of the following reasons:

• The primers are right but are not close enough together on the template and so don't amplify. Try designing new primers that are closer together or try a nested PCR protocol (PCR is done in two steps with different primers).
• The predicted melting temperature (Tm) for one or both of your amplicons is over 60°C (the highest Tm possible for DNA polymerase is 72°C). This can happen if there's too much GC content at either end of your amplicon; check this by looking at its G + C percentage on ea2bio.co

If you are having trouble amplifying your DNA, make sure that it is not old or contaminated. Also try using more of the PCR product as a template (100x).

If you're using a genomic template, make sure that it is clean and free of contaminants. A DNAse enzyme can be used to degrade any contaminating nucleic acids that are not part of the desired DNA.

Also, fresh, clean template DNA is always better than old or frozen samples. If you have access to an original tissue or cell line from which your product was derived (plasmid or isolated genomic DNA), use this for PCR amplification instead of using a PCR product as template in subsequent reactions.

If none of these things work, consider trying another protocol such as nested PCR (for more information on nested PCRs see [2]).

The cDNA synthesis step has failed to work. Repeat the cDNA synthesis step.

If you’re having trouble amplifying your PCR product, double-check the cDNA synthesis step by running a control reaction with no reverse transcriptase enzyme present. This will tell you whether or not your reverse transcriptase is working properly. If this step fails to work correctly, repeat it until it does so successfully.

After this step has worked correctly, check the purity of your RNA by running a gel to check the size distribution of the products produced during cDNA synthesis. If this step fails to work correctly and produces poor-quality cDNAs that do not amplify in PCR, repeat it until it does produce good-quality cDNAs that amplify well in PCR.

Glycerol may inhibit the enzyme. Make sure to run control reactions with no enzyme added, testing for contamination by RNA.

In a friendly tone: If the problem persists, you may want to check for contaminating RNA by running a control with no reverse transcriptase enzyme present. If your sample contains DNAse, RNase or inhibitors in template, you can use the proper negative controls to ensure that it's not inhibitory. Finally, if none of these seem like they could be causing your problems, consider checking your PCR reaction mixture for glycerol (this is often added as an antifreeze).

If a PCR product does not amplify, there may be a problem with either the reaction mixture or the machine itself.

• If a PCR product does not amplify then there is likely a problem with either the PCR reaction mix, the PCR machine itself or the sequence of the primers used to generate that product.
• The thermal cycler may be malfunctioning or have been used with too many different samples. This can cause cross-contamination between samples and lead to amplification problems.
• Contamination by previous products can happen if there was no clean-up step in between reactions or if reagents were reused from previous reactions (e.g., master mix).

Conclusion

If the PCR product doesn't amplify, there could be a number of reasons. It is important that you understand how to troubleshoot this problem so that you can work towards getting it resolved.