Hi all,

I've been working on a specific set of primers for the last couple of months. Previously I was getting a VERY faint band on an 2.2% agarose, just in the region of my required product.

I decided to further optimise my PCR by trying to increase some primer conc, dNTPs and also TAQ.

I increased my volume from 25 to 50microL.

Since i increased my volume (obviously keeping the same concns), i have discovered that neither my faint products nor an excess primers show up. Prmers show up only in the CN and the diluted DNA of 1:100.

Could it be that the reaction is taking place, and that is the reason that my primers disappeared?

If so, how come my product is not showing?

Is there a way how maybe i can increase the efficiency of ethidium bromide in agarose?

Regards

i bet your PCR product is very small since you are running a 2.2% agrose gel.
then you put EB in your gel, and that is what I will not recommended, since your DNA goes to the anode while the EB goes to the cathode, your small DNA may probably miss the EB and will not get stained. An after-run stain or put EB in the running buffer will be good.
to check this out, stop your electrophoresis before the front of the dye gets the middle of the gel, you should be able to see your primers (if over amount used) and DNAs (if any PCR products).

let me see if i understood correctly...

your suggesting that i run my pcr product over a 2.2% gell with EtBr, together with EtBR in the buffering slution as wel?