I have used the same primers for amplification and have been fine, but suddenly I started getting smear in both my negative control and test. I have checked all my reagents and NFW by using the same for other gene amplification and I have got positive results. I ordered new primers. But the same problem persists. I am still getting smear in my negative control and my test. In the picture below, the 3rd lane is the negative control and from 6th to 9th are my test. Kindly provide me with some solution.
A smear in the negative control lane of a PCR gel electrophoresis can indicate contamination in your PCR reaction. Here are a few possible reasons why this might be happening:
To troubleshoot and address this issue, consider the following steps:
- Strict adherence to sterile techniques: Ensure that you are working in a clean area using proper sterile techniques, including changing gloves frequently, using filter tips, and cleaning surfaces with DNA decontamination agents.
- Use of negative controls: Always include appropriate negative controls (no template controls) to monitor for contamination. If these show a smear, contamination is likely.
- Examine reagents and equipment: Check all reagents and equipment for contamination. Consider using new aliquots of reagents, and regularly clean and decontaminate equipment.
- Perform a contamination check: Run a control reaction using your PCR components (except template DNA) to ensure that none of your components are contaminated.
By systematically checking each potential source of contamination and implementing strict protocols to prevent it, you can reduce or eliminate the appearance of smears in your negative control lanes during PCR gel electrophoresis.
Thank you.
It does look like contamination. I would move to another researchers working area and use their pipettes,pcr mix water and plasticware but your primers and run 1positive and 3 NTC samples (water not dna). While this is happening strip down your pipettes and clean inside and out with soapy water. Clean your lab working area and get new plasicware. If the pcr is clean then move back to your area but still use the colleaguse pipettes and repeat the assay. If this is clean then repeat with your pipettes. If this is now clean you will ahhve solved your problem. When you get a smear like your gel shows this is often great over amplification of the bcr product which self anneals and forms long concatomers
@Paul Rutland Thank you for your insights. I have used positive control to eliminate any possible contamination with plasticwares or reagents. The 3rd and 4th lane is the positive control. It doesn't show any smear. Also, my concern is with the smear in the negative control (without any template). I think if that can be resolved then I will get bands in my test as well.
@lenin thank you. I have followed all the points that you have mentioned. But still I am getting dmear in the negative control
if ur getting result with other primer defnitely it shud be primer problem and check whether like wells of pcr instrument u were using.
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