Troubleshoot PCR smear problem - all I got was smear!! (Aug/18/2004 )
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.
Hmm, perhaps along with your "very very weak band" you also amplify the weak smear so you get a bright smear?
I have no other idea at the moment. Did you purify your DNA after the first PCR?
Did your ligation work?
What Thermal Cycler do you use?
You might have trouble with the thermal performance of the instrument. Lowering the denaturation temperature to 92°C might help.
maybe your template-DNA is somehow bad?
someone in our lab also had problems doing a "simple" genomic PCR (on Rice) and just got smears for all the reactions!
After extracting new DNA everything worked fine..
I was wondering if anyone has answers as to why my PCR product is one big streak from well to end. Somtimes a band shows up where expected but in the background (or foreground) is the large, sometimes bright streak. We've been having this problem for a while. Sometimes it's not there but nothing different was done with extraction or PCR parameters. We use optimal thermocycling conditions for our primers, so we're pretty sure it's not in the PCR reaction.
We've also encountered bright bands in wells followed by those streaks of some samples. Any suggestions would be greatly appreciated!
Thank you in advance.
I have had this problem before in the lab. But we realised that it was because People in the lab were too lazy to change the TAE buffer that was used to run the gel.
Hope this helps!
Thank you for the reply Kiwi.
We change our TAE every 5 runs. I'm one of the few in the lab so I have good tabs on how often it's changed.
We were thinking it may be carry-over contamination. How would that produce such intense streaks?
The only thing I can think of to produce long smears is chromosomal DNA from bacteria (what I'm used to working with). I don't know what your template is but, if there is too much of it, that will show up on a gel. I had a no-result PCR problem for the longest time so I got used to tweaking all the variables. My other suggestion would be to get new stocks of your individual components in case one somehow got contaminated. I am also assuming you follow storage reccommendations for the components and use sterile equipment, tubes, and whatever. Let us know what happens.
The template is mycorrhizal DNA. We deal with tiny root tips with a tiny amount of fungal DNA on it so we know that too much DNA is not the issue. We often encounter the problem of no bands which I've heard is common with this type of tissue but the smears seem to be interferring with amplification it seems because the occurence of bands is even less now.
I'm in the process of receiving new PCR components, have made up fresh solutions for extractions and use sterile tips, tubes etc everytime (always have) so we will have to be patient. Thanks. Will keep you posted.
Check if you have the same buffer in the made agarose gel as you use when running the gel...