I was undertaking a PCR reaction using a potentially large amount of ss-DNA following a SELEX procedure. I observed a smeared band of DNA. I have read that a large amount of template DNA can cause this but why?
If the gel is overloaded you basically get a "traffic jam" and the DNA can't migrate properly. Try loading less, or alternatively running the gel slower can give cleaner bands.
Dear Eleanor,
The PCR can amplify even a single template strand, so if you still get a smear after using lesser template DNA, there can be multiple reasons to it:
- there may be other impurities other than DNA in the sample you are loading
- try running a higher agarose concentration gel
- try running it slower at ~ 80V
I think you PCR product may form duplexes with you large ss-DNA templates. I have experienced this with denaturing acrylamide gel. I think in agarose gel the duplexs will form even easier, which migrate faster in the gel.
Why don't you send us a picture to reduce the extent of speculation in the answers?
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