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In this article we provide a step-by-step guide on how to assess and interpret electrophoresis gels after running PCR assays.
This resource is intended as a set of suggested steps for complete beginners and experienced users alike, taking the user thought the entire process from the first examination of a run electrophoresis gel, to a thorough analysis of any results shown.
By the end of the article you should have a better understanding, or refreshed your memory, of:
Understanding and interpreting the results of PCR experiments using gel electrophoresis is an essential skill for anyone involved in PCR work.
Gel electrophoresis is a technique that allows:
For a background in gel electrophoresis itself, and how to do it with Bento Lab, you can read our Introduction to Gel Electrophoresis.
Interpretation of electrophoresis gels is a very important step because it is usually the primary means of evaluating PCR results, either as the final step in the experimental process, or as the last low-cost step before proceeding to more expensive sequencing procedures. When done well and with appropriate controls (see our article on Controls in PCR and PCR assays with Bento Lab), it is also an essential tool for troubleshooting issues with your DNA extraction, PCRs and workflows.
The first step in assessing electrophoresis gels should be to record a good digital image of the gel. Digital images allow closer inspection of very small gels; image enhancement; annotation; and storage as part of a permanent experimental record.
To record a good digital image, you can:
It is always a good idea to make thoroughly documented records of experimental results. Consider pasting images into an electronic document (e.g. Google Docs or Slides) or printing out images to glue into a physical lab book. This will allow you to annotate the gel image with sample codes, results and interpretation, as part of your recorded experimental process.
Before documenting the gel, you can adjust the gel to make it clearer and more easy to interpret. You could use phone or desktop apps to crop and rotate images, and adjust image contrast, colour saturation, and gamma intensity. You could also invert the image to a white background with dark/black bands to make some gels clearer.
The physical quality of the gel can have an enormous impact on the quality of the gel image. Most physical gel issues can be observed directly after the gel is cast, but some issues are only visible after running the gel with amplicons and DNA ladder. Because of this it is always useful to quickly check the gel images for the issues in the table below.
The quality and extent of run for the DNA ladder can be used as an initial estimate of how well the gel ran overall.
The first and last lanes containing DNA ladder should have clearly visible banding in a ladder pattern, and should have run at least through the entire top half of the gel. These indicate that the gel has run successfully to the desired extent. For clearer band separation the gel can be run for longer or for the full extent of the gel.
DNA ladder bands should be as clear, distinct and crisp as possible. This may not always be possible across the full range of the ladder, but bands in the size ranges of the target amplicons should be well separated.
Poor running of the DNA ladder (e.g. faint or smeared bands, crooked running, poor separation) will indicate that you may get the same phenomena happening to your amplicons. This may or may not be a problem for your particular purposes, but it is not ideal in any case.
Smearing can be caused by:
It should be noted that there are some limitations in the resolution of bands in minigels compared to those in substantially larger gel electrophoresis units.
Step 4: Understand the DNA ladder
The DNA ladder allows the user to estimate the length of amplified DNA fragments in nucleotide base pairs by comparison to adjacent DNA ladders run at the same time as your PCR products.
The 100 bp DNA ladder contains a set of predetermined DNA fragment sizes ranging from 100 to 1000 in 100 bp increments, then (in some ladders) 1500, 2000, and 3000. Each band is provided in a particular concentration (ng DNA/unit volume). Consult the product information for your DNA ladder for specifics on the band size and concentrations.
The smallest bands are at the bottom of the gel (smaller DNA fragments run through the gel more quickly than larger fragments), the larger bands are at the top.
To estimate the size of your PCR amplicon, you can plot an imaginary line to the right or left of your amplicon and see how far it is up the ladder scale. For example, if it is half way between the fifth and sixth run on a 100 bp DNA ladder, then the size of your amplicon will be around 550 bp.
For best comparability of ladder to samples, you can run DNA ladder in the first and last wells within a gel, or adjacent to any PCR products if only a few are being examined. A middle lane of ladder could also be run to detect any differences in migration rate at the middle of the gel compared to the edges. This will help you detect any abnormalities in the straightness of the gel run and to allow better size estimation of amplicons.
You will hopefully have used a negative PCR control in your PCR to detect contamination in your PCR reagents or workflow. This lane should be blank. You can read more about controls in our article on Controls in PCR and PCR assays with Bento Lab.
If a band is present in the negative control (other than residual primers at below 100 bp in size) then your PCR mix or workflow was contaminated. If the band is faint and is not evident in other PCR products this may not be a major issue, but you should carefully check your reagents and workflow for PCR contamination and decontaminate as appropriate.
The above example shows a very faint positive result in the negative PCR control during a lichen barcoding exercise. This indicates that some of the results of this experiment may not sequence well and may need to be repeated. Specifically, any bands of the same size in other lanes may indicate that those PCRs also amplified this contaminant sequence.
If you are using a positive PCR control (recommended if you are encountering PCR failures, or for routine use when possible), you will have included a DNA template from a known sample that produces a known result when run. Check that the known result is present in its expected well, and that it is a clear bright amplicon.
If the positive control failed, and all your sample PCRs failed, then that suggests an issue with the PCR.
For more information on controls, you can consult our article on Controls in PCR and PCR assays with Bento Lab.
Before you began your experiment you should have had some expectation of the results of your PCR assay, for example a single amplicon band of a given size, one or two amplicon bands of different sizes, or a range of different amplicon band sizes. For example, in the gel image above from a lichen barcoding exercise, amplicons were sizes between ~500 bp and ~1100 bp. Consult the protocol for your PCR assay for more information of what to expect.
At least some, and hopefully most, of your sample lanes should contain amplicons corresponding to expected results. Different samples may produce amplicons of different sizes for the same target regions, depending on your specific PCR assay.
You may also see the following artifacts:
See our resource on Troubleshooting Non-Specific Amplification for more details of the above.
If you have no successes at all, then you can use your positive control to identify if it was a systemic PCR problem. If the positive control failed then there was probably a PCR issue. If the positive control was successful there was probably a DNA extraction issue with your samples. See our resource on Controls in PCR and PCR Assays for more information.
If most of your samples worked as expected but some didn’t, then the ones that didn’t may not have amplified due to either insufficient template DNA present or PCR inhibition caused by residual PCR inhibitors from the DNA extraction. If most of your samples were of the same subsample size and in good condition, then PCR inhibition may be more likely, and the extract may need dilution before use as a DNA template in PCR.
When assessing your PCR results, you may notice that the PCR products and DNA ladder do not always run perfectly evenly across the gel, and can run slightly askew or with the middle lanes running faster than those at the edge.
This is in part a limitation of small mini-gel tanks and gels, and it may be more pronounced if you run your gels faster than recommended. Better quality gels may be achieved by running the gel at a lower voltage for a longer amount of time.
Crooked running of gel lanes can also be caused by gel tank electrodes becoming misaligned. If this occurs then check them and straighten them if possible, taking care not to damage them.
If you are expecting multiple bands in your electrophoresis gel but only see one, then you may be experiencing some form of PCR bias or amplicon dropout.
PCR bias is a phenomenon that can occur whenever multiple amplicons are targeted in a PCR but they exhibit different amplification efficiencies. For example, this may result in an expected two bands on a gel being visible as only one band, or as one very strong and one very weak band.
A similar phenomenon that can result in missing bands is called allelic dropout. This is a phenomenon where two gene variants (alleles) are being amplified from the same organism in the same reaction, but one fails to amplify, resulting in a false result of homozygosity. The possibility of allelic dropout is an important reason why PCR-based medical diagnostics make extensive use of multiple controls and standardised procedures.
Well-designed PCR assays are experimentally optimised to reduce PCR biases before publication, and so provided the protocols are followed precisely then this should not be a major issue for the majority of assays.
If your gel is showing unexpected numbers of amplicons (for example double bands in single band PCRs), or smears or other unexpected results, then your PCR may have suffered from either contamination or non-specific amplification.
You can compare additional bands in your results with your negative control to see if there is systemic contamination. For example, in the example above, there is a trace contaminant in the negative control (NC) that is possibly showing in PCR products 1Dp and 2Dp.
If any double bands are not caused by systemic contamination, then you can then consider the most likely possible causes. In the example above, the most likely explanation is co-amplification of additional fungal species present within the lichens which were being barcoded.
For more information on non-specific amplification you can read our article on Troubleshooting Non-Specific Amplification with Bento Lab.
Interpretation of electrophoresis gels is a very important part of assessing the results of your PCRs, and detailed inspection can provide a lot of information about your results, and what is working well or badly in your experimental method.
By examining your electrophoresis gels in a systematic, step-by-step process, you can:
This should help you assess your PCR results with more confidence, identify problems, and produce reliable results in your experiments!
Please let us know what other resources, advice, and tips and tricks for using Bento Lab that you would like us to produce in the future!
