Troubleshoot PCR smear problem - all I got was smear!! (Aug/18/2004 )
have you ever tried polymerase for long PCR, i think your template (1.5K) a bit long for ordinary EXTaq.
I have a similar problem.My target fragment is 3 kb from chicken brain cDNA.My PCR produces only smear from the beginning to the end of the well!!!!and I can't see any bands:(( Mg conct. of the reaction was 1.5 mM. I increased it up to 3 mM and this time smear disappeared but ı can't still see any bands!!I also change the annealing temp. and finally try touchdown PCR but nothing changed:(((
Any comments?? Thank you...
i used to face this problem. Please try to increase your initial denaturation time at 95'C for 5-7 mins or do the hot start to make sure that your template is completely denature. This works to me..
Good luck and all the best
hii, i think its very basic problem,check the mg level in your PCR and try to increase annealing temperature for 2-3 centigrades,second one you should column purifirs RNA/DNA beyond trizol or chemically purified DNA/RNA.you will get the result byee n best of luck
national institute of virology
It sounds that as a conclusion you could say, that everything could be the problem .
I had smear after after changing the temperature profile to a lower annealing temperature. After readjusting the temperature ist worked fine again.
But as I said, it could be nearly everything .
Does anybody ever doubt the primer itself? I have had a PCR problem before. It turned out that the company gave me the wrong primer. When I resynthesised the prime, promlem solved.
Well, if you have an established PCR that always works (or almost always) and you encounter a problem and you can not solve it by replacing your reagents, have tried it on different samples, cleaning everything thoroughly, not getting positive controls to work.... I'd definately considers the primers. It's just not the first thing I would check.
The main problem with your case is the amount of template DNA. In case of higher amount of DNA PCR usually fails or you got the low yield of the desire product. You should standerized the template DNA.
Further if this do not work, you can also try various concentration of DMSO, SDS with low annealing temperature.
I was absolutely struggling for weeks with a PCR from tobacco genomic DNA.
the target fragment is 1.6kb, the primers match the target perfectly, both of them have a Tm 58. polymerase is Takara ExTaq. gDNA is well coz the positive ck always works well.
I got one "very very" weak band around 1.6kb(no other band), with some weak smear (94C, 1min; 48C, 2min; 72C, 2min; for 36 cycles.). increasing the anneal temperature(even just 50 or 49C), or decreasing anneal time(1min or 1min30s), there were no band. In lower anneal temperature, all I got was smear. The single primer PCR results showed some nonspecific bands but all of them were not around 1.6kb.
Coz the band is too weak; I try to do a 2ed PCR to enrich production, then I can digest the fragment with some RE to confirm it. I used a final 1:200 dilution of 1st PCR production as template, same primer of 1st PCR, anneal tm I tried was from 50, 52, 54...to 60C, 15 or 20 cycles. All I got was smear. What’s wrong??
I have done a ligation of the 1st PCR fragment today, if it works I can confirm it very easy. But I'm just wondering why my 2ed PCR doesn’t work????
Thanks a lot.
Hi PCR people, I also have a smear bands problem, or not any band at all and just the smear. The strange part is that just one or two months ago, I was having very clear bands. My samples were stored in RNAlater at -20, so I can't believe that they are completely degraded. I'm extracting the DNA with a Phenol based technique, by the way. What I wonder, and I don't know if it might have happened to anybody, if it is that a small degradation of the samples, might originate small fragments that can compit with my primers for the amplification. Any comments? Thanks a lot.
what i suggest you should set three parallel reactions. In one reaction just increase tm by 2-3 degree. In another one increase tm by 2-3 degree with DMSO (1-10 %). In third reaction simply put DMSO (1-10%). Mostly 3-% DMSO conc. is effective.
it may solve your problem or you would find root cause of problem.