Why Smeared and multiple bands occurs in polyacrylamide gel electrophoresis while running ssrs product amplified by touch down PCR?

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Michael J. Benedik

Seems to me that your lanes are overloaded. Try the gel again with much less of each sample (maybe 10% or 25% of the loaded sample). Or use a gel with larger lanes for each sample.

Alex Ignatov

Sai Timmarao Koka It looks like you have different template DNA concentration/purity. Each sample must be adjusted to get equal PCR product concentration.

Paul Rutland

I would run the gel at a lower voltage.The smiling bands could be caused by too much salt in the sample or by being run at too high a voltage. I agree with Michael J. Benedik that some of the samples are overloaded.

Ljubiša Veljović

Try to put lower voltage and dilute samples at least 1:10. Your electrophoresis is to "fast" according to picture.

Nikolay Klyashtornyy

There could be several reasons why you are observing smearing and multiple bands in your polyacrylamide gel electrophoresis (PAGE) while running SSRs product amplified by touch-down PCR. Here are a few possibilities:

Incomplete denaturation of the PCR product: If the PCR product is not completely denatured, then it will not migrate as a discrete band on the gel. This could result in smearing or multiple bands. To ensure complete denaturation, you can increase the temperature and/or time of the denaturation step.

Non-specific amplification: If the PCR primers anneal to non-target DNA sequences, then you may see multiple bands on the gel. To prevent this, you can optimize the PCR annealing temperature and/or the PCR primer concentrations.

Contamination: Contamination can lead to the amplification of non-target DNA sequences, resulting in multiple bands on the gel. To prevent contamination, make sure to use sterile techniques, and separate the PCR set-up from the post-PCR analysis.

Problems with the gel: If there are problems with the gel itself, such as uneven polymerization or buffer depletion, then this can result in smearing or uneven bands. Ensure that you are using fresh gel and buffer, and check that the gel has polymerized correctly.

Variability in the SSR loci: SSRs are known to be variable in terms of length, and this variability can lead to the observation of multiple bands on the gel. To minimize this variability, you can try to design PCR primers that target the most conserved regions of the SSR loci, or use a different PCR strategy altogether.

Troubleshooting the issue will require careful examination of your PCR conditions and gel electrophoresis protocol. By identifying and addressing potential sources of error, you should be able to obtain clearer bands on the gel.

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