PCR band too thick - PCR troubleshooting question (Mar/30/2010 )

Hi,

This is probably a stupid question, but I've been trying out different primer combinations to pick the one with the "cleanest" visualization of my 120-160bp product on a 2% agarose gel.

So far my bands have been thick and each lane has a faint glow thoughout. I was wondering what i can adjust to make cleaner bands. The lane with the ladder is pretty clean... does that probably mean it's a PCR prep issue and not a gel set up issue?

I know too much DNA or Mg can create too much product and smear the bands, but I'm not sure where is a good place to start adjusting things. Right now I'm using 1.5mM concentration of Mg and 500ng genomic DNA. How much less MgCl or DNA should i be using?

or is it probably something else... I'm using 1.0uM of each primer, 0.8mM dNTP, and 0.63U Taq

any thoughts would be great!

If your gel is giving you nice and clean bands for your size marker, of similar size to your PCR product, then likely your issue is with your PCR product.

This can be due to a number of issues, including your PCR assay itself. If your primers are amplifying some non-specific fragments, or even if your PCR product has a DNA sequence that lends itself for some heterogeneity, then you will get some fuzziness when you run your PCR product on a gel.

My best advice to minimize this effect is to optimize your PCR assay. Reduce the amount of primer you are using (1.0 uM of primer is quite a bit; you should be able to easily half that), increase your annealing temperature, reduce MgCl (1 mM is typically more than enough, but then again that is assay specific).

Ultimately though, be aware that sometimes no matter what you do you will not be able to make those bands any sharper.

Good luck

Also make sure you are not simply overloading the amount of DNA in the lane. Too much DNA will smear no matter how good the PCR reaction is.

^^ Above is true. starting template can be lowered and you should see a cleaner band.

Or just load less of the product to the gel.
I typically run around 2ul of a 25ul reaction to check, but if its too bright and i need a good picture I'll use less....

leelee on Mar 30 2010, 04:17 PM said: