Since its invention in the 1980’s, PCR has become a cornerstone technique in molecular biology, with research applications ranging from cloning to gene expression analysis, and clinical applications including genotyping and infectious disease diagnosis. PCR is great when it works, but can be painful to troubleshoot when it doesn’t. Although PCR reactions that yield low amounts of amplicon, off-target amplicons, and/or primer dimers won’t leave you jumping for joy when you run your gel, the most painful PCR reaction outcome is one that produces no band at all! Here are our top tips to help you troubleshoot a no-band PCR.
Since its invention in the 1980’s, PCR has become a cornerstone technique in molecular biology, with research applications ranging from cloning to gene expression analysis, and clinical applications including genotyping and infectious disease diagnosis.
PCR is great when it works, but can be painful to troubleshoot when it doesn’t. Although PCR reactions that yield low amounts of amplicon, off-target amplicons, and/or primer dimers won’t leave you jumping for joy when you run your gel, the most painful PCR reaction outcome is one that produces no band at all! Here are our top tips to help you troubleshoot a no-band PCR.
1. Have You Forgotten Something?
It might sound too obvious, but a common reason for no PCR bands is the omission of a component from the reaction. It’s easily done, but it’s fortunately also easy to troubleshoot by using a checklist. Write down every single component that should be in your PCR reaction, and tick each one off as you add it to the tube. In addition, make sure that you are using the components at suitable concentrations by consulting the PCR polymerase manufacturer’s guidelines.
Tip: besides saving you time and reducing potential pipetting errors, using a commercial PCR mastermix will also reduce the risk of omitting some of the PCR components.
Standard PCR Reaction Components:
- PCR Buffer – this is often supplied as a 10 x concentrate but check to make sure this is the case!
- dNTPS – added either as a combined mixture or as 4 individual components (A, T, C and G)
- MgCl2 (if not already included in your PCR buffer)
- Forward Primer
- Reverse Primer
- Template DNA
2. Is Your Template Present in the Reaction at All?
Double-checking that you’ve added something from the tube labelled ‘template DNA’ to your PCR reaction may not always be enough! Bear in mind that that the DNA preparation you are using may not actually contain your target sequence at all. There are several situations where this may happen. Here are two examples:
- If you are amplifying from a cDNA template, you will only get a target amplicon if the corresponding gene is expressed in the conditions the cDNA originates from. Including a genomic DNA control when running PCR reactions with cDNA templates will help you pinpoint whether the issue lies with the cDNA or some other component of the PCR reaction.
- Your target sequence might also be missing from your cDNA if you exclusively use oligo dT primers to reverse transcribe mRNA, since oligo dT primers bind polyA tails at the 3′ ends of mRNA transcripts, and thus exhibit bias against 5′ ends. You can get around this possibility by using a cDNA synthesis kit that is optimised for complete 5′ to 3′ RNA sequence representation, for example, Bioline’s SensiFAST™ cDNA Synthesis Kit (contact us for a free sample), which contains an optimised mix of random hexamers and anchored oligo dT primers for unbiased mRNA priming.
3. Less Is More – Dilute Your Template
PCR inhibitors may originate directly from your sample, for example, certain fungal and plant secondary metabolites, or may be introduced during sample processing or nucleic acid extraction, for example SDS, phenol, chloroform, and other detergents or organic solvents.
The presence of inhibitors can interfere with PCR in a number of ways, resulting in everything from a low-efficiency PCR to no amplification at all:
- By binding nucleic acids and/or polymerases and interfering directly with DNA replication, for example, by preventing primer annealing
- By sequestering essential co-factors such as Mg2+. EDTA is a culprit here, so bear this in mind if you are eluting your DNA in a solution that contains EDTA (e.g. TE buffer).
- By denaturing the DNA polymerase rendering it useless (e.g. SDS).
Generally, inhibitors are more likely to be a concern when using homemade DNA extraction protocols, and most commercial DNA extraction kits yield PCR-ready DNA. However, inhibitor removal is not always guaranteed, especially if you are working with certain sample types, including, but not limited to, blood, faeces, and soil.
Before you fork out for PCR inhibitor removal kits, try diluting your DNA template 10-fold in water. Remember that PCR amplification theoretically only requires one copy of the target sequence, and by diluting your template you will also dilute any inhibitors that may be present. If you suddenly see a band or a stronger band than before, you can be confident that inhibitors are an issue. If diluting your PCR doesn’t give you the whopping PCR band you need to proceed, try cleaning up your DNA with a PCR inhibitor removal kit, such as Zymo Research’s OneStep PCR Inhibitor Removal Kit (contact us for a free sample).
4. Watch out for Surprise Introns
If you are amplifying genomic DNA from a species whose genome sequence is not fully annotated, your target region may contain unexpected introns that can greatly affect the outcome of your PCR, particularly if these increase the size of the region substantially.
If possible, check whether the corresponding target region in closely related species is annotated to have introns, and optimise your PCR cycling conditions accordingly, i.e. increase the extension time.
If there are no other genomes sequences available, it might be worth isolating RNA and performing PCR on the resulting cDNA, where any introns will have been spliced out.
5. Have You Missed Any Primer Issues?
When it comes to PCR troubleshooting, primers can be the source of much frustration. There are many things to consider BEFORE you even attempt to run a PCR reaction, but if you’re reached the no-band point and you suspect you haven’t been thorough enough with your primers, it is worth revisiting this topic.
Primers are a whole chapter by themselves, and we always recommend following guidelines provided by your PCR reagent’s manufacturer, at least to start out with.
Here’s a few other tips to help you along:
- Check that the sequence of the forward primer is identical to the target’s 5′->3′ first strand sequence, and that the sequence of the reverse primer is a reverse complement of the target’s 5′->3’second strand sequence? If not, your PCR will never work!
- Design primers that are free (as much as possible) of secondary structures. Most primer design programs will predict secondary structure formation (e.g. primer dimers and hairpin loops). You will usually see primer dimers migrating as low molecular weight bands on an agarose gel.
- Although it’s usually possible to predict a suitable annealing temperature based on primer melting temperatures, this strategy won’t necessarily give you the optimum annealing temperature, and in some cases it may not work at all. Run a gradient PCR to determine the optimal annealing temperature. Most PCR machines have a gradient PCR function, all you have to do is set the upper and lower temperatures and the increments between each. As an example, you could start at 50 °C and go up to 60 °C in 1 °C increments.
We realise that PCR and PCR troubleshooting are huge topics, and we have tried to focus our tips on those most likely to help the majority of researchers. If you have any other tips you’d like to share with us, drop us a line in the comments section.
If you’re stuck with a no-band PCR and would like support, get in touch with any of our team at firstname.lastname@example.org. We will be more than happy to help you!