Hello everyone,
Lately I am having trouble with PCR of my DNA samples. I keep on getting a band in my negative control. My targeting region is very small (190 bp) on the 18S mtDNA. This band is slightly smaller or the same length. Primer clouds are also visible at the very end. I first thought of contamination, that is why I changed everything, pipetting tips, reagents, tubes, cleaning with bleach, but still a band. Two strange things rising up, it worked in the very beginning, also the blocking primer which does not seem to work properly the last PCR times. Plus if I add water as DNA template or the buffer used for the DNA extraction, the band shows up, but if I only use the mastermix for the PCR, there is no product at all visible in the gel.
PCR is running at 35 cycles, which is a high amount, but it used to work before and anneling temperature was set higher already. I am kind of desperate, maybe anyone does have a good suggestion what I might can try to solve this problem.
In any case I would be happy about some help!
Thanks in advance.
Hello,
You can try using the mixture without the primers and DNA template, if the band still appear; it means that there is contamination ( sometimes contamination comes from your procedure not only from the reagent). You can try increasing the denaturation time too.
You have post pcr contamination of a reagent,water, pipettes or working area. If you have a no template control that amplifies a product then it is always contamination,
borrow or buy all new primers and reagents, borrow pipettes from anyone else . Working in another persons area run a pcr with 4 no template controls and one positive sample. While this is happening thoroughly strip and clean your pipettes, clean your working area with plenty of soapy water and change all of your plasticware.
Contamination is hard to get rid of so a certain way of getting rid of it is to design at least one of your pcr primers outside of the current ones then the contamination will not amplify if this is possible.
To avoid it happening again be very careful opening plates and tubes ( aerosol spreading of pcr product) and do not bring gels or wetted gloves that have been in the buffer tank or picked up a used agarose gel back into your working area because these will spread contamination
agree with Paul Rutland
If the product is small, you should also consider the possibility of primer dimer. although you should also have it in the samples.
rRNA genes can also be amplified from bacteria, which can be found anywhere. Clean everything, use UV if available, use filter tips and separate pre- and post-PCR areas
In addition to confirming all the answers, you can change the pipette tips or pipette you use. If you are using pipette tips without filter sometimes DNA (or other contaminants) splash on the pipette. Thus, I strongly recommend use the filter pipette tips and clean all the pipettes you use. If you already use filter pipette tips, all you have to do is clean the pipettes and do the PCR with new sterile filter pipette tips.
It doesn't appear to be possible, but I've had this issue before.
If there is no band in a PCR sample without buffer and water, there is a chance of contamination from the pipette. Generally, we add template using 2µl pipettes. I think contamination is from the pipette used for addition of DNA template, water and buffer to the PCR tube. Try to clean the pipettes, use filter tips, or use separate pipettes for each (buffer, water and DNA) addition (just to find the source of contamination). Good luck
There are a few possible explanations for why a negative control in a PCR assay might show a band. Some potential causes include:
It is important to troubleshoot and identify the cause of the problem in order to obtain reliable results from your PCR assay. Some steps you can take to troubleshoot the issue include repeating the assay with fresh reagents, checking for contamination, optimizing the PCR conditions (such as the annealing temperature), and using different primers.
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