Smearing on Gel - PCR product shows up as smears on gel (Sep/03/2004 )
HI,
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.
kudna
Hi Kudna,
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!
Kiwi
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?
Kudna
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...
/Nina
yes, of course! I do have the same buffer in the gel as in the gel box.
Thanks.
Kudna
Your problem sounds like non-specific amplification to me, have you tried raising the annealing temperature and/or lowering the primer concentration in your PCR. Another trick would be to lower the DNA concentration in the reaction, sometimes too much DNA can cause these sorts of effects.
I agree with Bob1, but what is the theory behind smearing? Why do we get amplification of fragments of evenly distributed size which show up as a smear? Sometimes you just could not find a clue.
What concentration of Mg2+ are you using? Too much Mg2+ can cause smearing and decrease the specificity of the annealing. The optimal range is 1mM-2.5mM
Hope this is helpful
Ihab
p.s. check out this PCR troubleshooting site
http://info.med.yale.edu/genetics/ward/tavi/PCR.html