Hello, I am doing PCR on fungal DNA samples and use EF1 and EF2 primers and the New England Bio Labs Phusion Taq Polymerase. I have had successful PCR reactions but over the past month I have been getting a uniform smear in each well including my negative control. I still have nice crisp bands, but the smear is a problem because I am sequencing these products. I use filter tips and my prep areas for pre-PCR and post-PCR are separate. If anyone has experience with this or any advice, it would be much appreciated. I have attached an image of a recent gel, the first well beside the ladder (farthest to the left) is my negative control (PCR mixture with no DNA, ultra pure water replaces the DNA). I have changed out my reagents/water and the smearing has not stopped.
Some prevalent errors might cause the PCR product to get bogged down below:
- Misread gel: If the gel is not poured correctly, it will not polymerize or harden uniformly so that the molecules smear.
- Well overloading: If the wells are too much packed, or the sample isn't diluted appropriately, the surplus sample might be smeared through the gel. Moreover, when the gel is shifted after placing the selection well, the model might break down. This might be spread as well.
- Improperly Prepared Sampling. Divides into an enzyme that cuts the molecule in particular areas to prepare the protein or nucleic acid sample (restriction digestion/). If not correctly performed, the enzyme might uncontrollably break down the molecule too much and cause a smear. Moreover, the wrong buffer or temperature choice might lead to an inappropriate enzyme function, producing flushing, and not most minor.
- Pollution: if a sample of DNA is polluted by a protein, which can also produce smear.
This is a relatively common problem for agarose gel electrophoresis, and there can be plenty of information elsewhere.
Good luck!
Mostly smearing caused by degraded DNA which occurs during the sample handling. Handling can be improved by slow pipetting, avoid major temperature fluctuation to avoid DNA degradation.
The fact that you get the smear in the "no DNA" control suggests that there may be some contamination in your PCR reagents by a DNA that does not amplify or amplifies non-specifically, but very poorly with the primers that you use in the reaction. As to sequencking, did you actually try to sequence the reaction products ? It may be that the smear background is not too much of a problem: since the major product that you see on the gel is clearly way over background, you may expect that it is also going to give a sequencing chromatogram with strong peaks that will be clearly stronger than the non-specific background arising from the smear.
A possibility is that you still have enzyme stuck to the amplified dna. You could try adding SDS to your samples prior to loading to dissociate any dna.protein binding maybe to a final concentration of 0.05% but overall I agree with Pierre Béguin
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