I normally use a nested PCR on 16S-23SrRNA region, for diagnosis of a plant pathogenic bacterium. It is a well-established and almost “trivial” protocol, shared in many laboratories.
Recently when the nested PCR products are loaded on 1% agarose gel, smirs appeared instead of usual bands, even in blanks. This happens with samples previously checked by qPCR, for pathogen presence and concentration.
On the contrary, the products of the first PCR are better defined.
We tried to solve the problem by changing the different variables of the system:
primers working aliquotes, primers pair (different pairs can be used for the same genomic region and with the same amplification protocol), Taq Polymerase, dNPTS; thermocycler; TBE buffer, electrophoresis cell and power supply, agarose, DNA staining (new Gel red and Red Safe aliquotes); we also tried by lowing the template concentration, but nothing worked.
Other types of nested PCR on the same DNA samples, but on different genes, didn’t have any problems.
Could someone please suggest me an explanation for those bad results?
Can understand your concerns that is really puzzling, If I understood well the upper gel is the first PCR and the one below the Nested products is that correct ?
- The second gel looks like a lot of Genomic DNA contaminating but if that would be the case the first PCR should look the same so it's not that.
- This type os smear can be produce by excess of salts but there's one lane clean so again not the explanation.
- as for a nested PCR I wonder how many cycles you did since the first PCR already shows tons of products you're in risk of generating weird results
It may be be due to excess amount of protein in sample ... Plz... which method was used for extraction ?
Jean-Pierre Dangy, no, both images are nested PCR, the upper is the standard output, the lower is the current result; the DNA samples are not exactly the same, but protocols, reagentes, thermocycler etc did not change. As template for the nested PCR we use 1 ul of a 1:50 dilution of the first PCR; to test if the problem was the excess of DNA, we tried to use 1 ul of 1:100 dilution, but it did not work.
Yousif Mousa Alobaid Ahmed, we used a CTAB method for total nucleic acid extractions, from plants and from insects (slighlty different). But normally the nested PCR from both the matrices gave band like those in
the upper gel.
Did you run a negative control including all the reagents but no DNA ? This would show that the input DNA is not the problem. It could be due to a contamination of one of your reagents. you should also test both primers separately if possible just to avoid strange mispriming
Ok... Do you remember that your purification before loading your sample for nested PCR and give you normal range which is suitable for detection ?
This will seem strange to ask but is there any chance you recently changed your PCR tubes (plates, strips - whatever you're using)? A few years ago when I was working on a library for NGS I suddenly had a difference in my PCR products (fewer amplifications and smearing on the agarose gels) after having run over a hundred samples. After much trouble shooting (similar to yours) I finally realized I had gotten a new brand of tubes -same volume and same thin wall (supposedly). At first I really thought I was nutso for thinking that was the cause of my problems, but it actually was the reason! I used some tubes that I had used during the development of my protocol and everything came out as expected! I ordered the original types of tubes and finished the project. - for clarity, I had used individual 200 ul PCR tubes for initial testing and switched to using 8 tube strips afterwards with no issues. It was a change to another type of 8 tube strip that caused the problem. (There was nothing inherently wrong with the new tubes. I have since used them in other projects. I just never change tube type/brand anymore while in the middle of a project).
Good luck.
Because of the mixer of debris with your DNA/RNA. So, try to free from it by using a good kit.
Hello! I am a graduate student coming into a lab that has also been having this problem on and off for a while now. Lab lore seems to say that it tends to stop working in the summer and start again sometime in the fall, but this last year, it worked only intermittently over the winter. We have ruled out it just being a reagent problem. Our smeary gels look almost exactly like yours so all this to say, if you figure it out please let me know and also if we make any headway I will be sure to update you!
Well, I have exactly the same problem with a circularized RNA that looks great in the first gel, I cut the band, I purify it with a cleaning kit and I have the same result. I also changed the individual tubes that I used at the beginning, for some in strips, I will follow Molinero's advice and return to the individual ones.
If I find the solution I will tell you, please I ask you to do the same, regards.
you could dilute your product from the first PCR 1:100 and increase the number of cycles in the second. My problem is worse because I am trying to re-amplify the product of the first PCR.
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