One of my PCR products shows a weak band in gel electrophoresis compare to positive control. It is for detecting leukosis virus in blood. What does it mean? should I increase annealing temperature to fade that band or it means that it is positive? DNA extraction is same for this sample and positive control.
thanks
A weak band just indicates there is less DNA of that particular size. I would say it is positive, just with less starting template than the positive control possibly.
there are many reasons for that, below are some the reasons which I know.
1)The gell concentration was high may be more than 0.8.
2) The DNA samples are old preparation.
3) The DNA fragment was small.
4) The steps or cycles of PCR are not reasonable.
5) The running buffer has not had enough water to flood the gel.
6) The volumes of DNA and primer added to the PCR reaction were unsuitable.
increase template concentration and try gradient PCR once and standardized
Thanks, but I can see a sharp band from other sample in the same gel.
Then it might problem with that particular sample did you checked nanodrop for that sample? Try PCR One more time after knowing the template concentrations
If the weaker band is a different size from the expected band then you can try increasing the annealing temperature or run the pcr with added DMSO at concentrations of up to 8%. try 1%, 2% etc up to 8%.
If the band is the same size as the control sample but just weaker then it is because there is less template dna in your samples so run a few more cycles and use a more dilute positive control. Make sure to also run a no template control as a check against pcr contamination.
Here, I see great suggestions from other researchers. The lighter band of a gel is indication of less concentration of DNA. In addition to this, I think knowing the concentration of DNA before the PCR and make sure the purity of DNA will be helpful from Nanodrop. If DNA purity is not good enough that could be another reason for faint band because the DNA concentration was high enough to observe under the gel but it might not be the PCR product. Make sure your elution buffer to elute the DNA has no salt around the CAP. Starting DNA for control and experiment must have the same concentration and purity. May I know which stain do you use to observe the DNA? How sensitive is your DNA stain? please, make sure these parameters as well.
Here are some possible reasons for a weaker band:
Before adjusting the annealing temperature, it is recommended to optimize the PCR conditions to improve the amplification efficiency of the target DNA. This could involve adjusting the annealing temperature, optimizing the primer concentration, or extending the extension time.
If you have already optimized the PCR conditions and are still seeing a weaker band, it could indicate a lower concentration of the target DNA in the sample. In this case, you may want to consider re-extracting the DNA from the sample and repeating the PCR to confirm the result.
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