Hi all,
My PCR products always show a very faint smear spreading all the way long (from the wells to the band of interest). I´m not being able to get rid of it. I have the same problem with two different PCR reactions. I've checked the DNA quality by running it in an agarose gel 0.8%. It looks fine.
I've tried:
- Reducing the DNA amount: Then the intensity of the amplicon of interest decreases, the smear is reduced but is still present.
- Reducing the number of PCR cycles: Same (the intensity of the amplicon of interest decreases, the smear is reduced but is still present)
- Higher annealing Ts: The smear remains.
I'm using a NOT HotStart Taq Polymerase (Kappa). Do you think that using a different type of polymerase can make the difference?
Any other suggestions?
Thanks in advance
Hi Cella,
That is very normal. Frequently, we will see that phenomanon. There is no need dwelling on it. If you just want to check and see if a certain gene (or DNA fragment) is amplified, with the right-sized product amplified as shown, you should just move on. If you need the PCR product for downstream experiment and you don't want the 'faint smear' parts, you can just cut out the band and do gel-extraction.
Yes, strong protein binding to the DNA may be preventing proper gel separation. Try adding 1% SDS to the loading dye just before running the gel. We always use taq DNA polymerase from Thermo Scientific and we dont have such problem. May be try with a different polymerase to see if you still have the same problem. Good luck.
I'm using 60ng of DNA in a final reaction volume of 20ul (3ul of DNA at a concentration of 20ng/ul measured with Nanodrop).
Thanks, Nadine.
Sampath, Do you think the smear can be an artifact generated during the gel run? Would that mean that the smear does not necessarily reveal the presence of unspecific PCR products or excess DNA?
There's something that I´ve not mentioned and could be important: When looking at the gel using the GelDoc the smear does not appear. Is just by forcing overexposure, and modifying brightness, contrast,.. that the smear appears.
Thanks
I always used 20 ng of DNA in 20µl total reaction volume.
I think this page will help you: http://www.bio-rad.com/de-de/applications-technologies/pcr-troubleshooting#gel3
or as you aer getting your product after PCR reaction so you could go for PCR product purificationby using various kits available in market before running on agarose.. hope this will work
all the best
Hi Cella,
That is very normal. Frequently, we will see that phenomanon. There is no need dwelling on it. If you just want to check and see if a certain gene (or DNA fragment) is amplified, with the right-sized product amplified as shown, you should just move on. If you need the PCR product for downstream experiment and you don't want the 'faint smear' parts, you can just cut out the band and do gel-extraction.
You can just try to add extra MgCl2 to your mastermix and let me know what are your results look like?
Best,
Zack
Hi Celia
Indeed DNA concentration, DNA quality and non-specific amplification is the major factor for the smear formation. Appart from that you should consider using the fresh buffer. Because i had this experience once and findout buffer is the cause for smear.
Best luck
sam
You can decrease the amount of used primers
its mybe reduse the smear.
Hi, i think it is a high concentration of DNA template , it is no matter and it is not affect your results, good luck.
PCR set-up depends on the amplicon size. hence, look for appropriate set-up for elongation step. also u can use as low as 10ng input DNA it should not matter.
Smear is basically an indication of primers binding to nonspecific regions and amplifying. As specified by Shirzad Fallahi you can reduce the primer concentration.
I have had that problem, but I soon realized that it may be due to many contributing factors:
As for my case, I troubleshoot one factor at a time until i found out that my root cause was the number (1 and 2).
Hope this helps
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