Why cDNA negative-controls are showing bands in a gel after qRT-PCR is done with a housekeeping gene?

Hello i am trying a pair of new primers. Unfortunately I do not have enough reactions to perform a temperature gradient.

My HK primer has Tm of 60.3 (forward) and 60.8 (reverse) and the other primer I will use has 60.4 (forward) and 58.9 (reverse).

However it is also stated in the data sheet that my primer with 60.4 Tm has a strong secondary structure formation. I have seen that even though standard PCR kits have some amount of DMSO it still can be useful to add DMSO in order to reduce the Tm.

If so would you recommend additional DMSO and is 5% is a good starting point?

Also in this case can I blindly predict my Ta by subtracting 5 degrees from the lowest Tm I have?

Any help would very much be appreciated. :)

Best Regards

Hi,

I have done cloning for years and just recently I have encountered this problem. When I do my PCR screening with Taq, I never run along a negative control because I know what size I am looking for. However, this time, I am looking for a band at 158bp. I am afraid that cannot tell the difference between PCR product and primer dimers, so I run a negative control along it (template that primers could not amplified). I saw a band exactly the same size as positive control.

So, I did it again without any template and I still got a band same as positive control. So I changed my water, buffer and make fresh primers. But I still have the same result.

Are those just primer dimers running the same size as my PCR product? My primers are 25 and 27 bp. If those are just primer dimers, how can I tell the difference from my PCR product? I am running it on 2% agarose gel and I used 100bp DNA ladder.

Thank you.

Regards,

Wan Ling

I have added everything except template DNA. However it is still showing a thin band on gel. Is there any solution to this?

I am doing my undergrad thesis that requires me to do PCR-RFLP analysis on DNA that I extracted from some blood samples. Initially, I was using the Qiagen mastermix for the optimization of my entire process using just 6 samples out of 100 samples. I successfully completed my optimization step, and got my desired results for those 6 samples. Once I have the measurements of the entire process i.e the amount of mastermix, forward priner, reverse primer, DNA template, and nuclease-free water along with the optimum annealing temperature for my selected primer, I proceeded on to doing the exact same thing on the remaining 96 samples serially. But I started encountering an unexpected error. For some reason, I started getting a faint band in my negative control(which only has nuclease-free water and no DNA template). At first I thought any of the reagents were contaminated, so I conducted a series of PCR reactions where in one I would use a freshly new mastermix, another time a fresh made working solution of primers and also an intact nuclease free water. In every PCR, I am getting a band in my negative control. Today I also tried using a mastermix of an entirely different company i.e. Takara Bio, but got the same results. I am just frustrated at this point. Can anyone help me figure out what is happening? How can I troubleshoot this and complete my thesis!

I want to do PCR amplification with my full-length gene and the addition of P2A fragment at the end of my gene. However after doing PCR, I run gel electrophoresis for analysis. But it doesn't include the band for my gene. I run the same template DNA with other primers for smaller fragment, and it has the band. I tried to redesign my primers for full-length fragment, but it still don't have the band for my gene. Can anyone help me? Thank you

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Good day everyone,

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