Detecting 12 nt differences in a 600+ bp pcr product - (May/17/2005 )

Hi -

I am working on a protocol for detecting 12 nucleotide differences in a 600+ bp pcr product. Looking for any advice (lol doing something else is not going to work). We can 't use fragment analysis since our product is too large (and yes we need it all). I have so far had very limited sucess with using Metaphor agarose (2% to which we add Synergel (a synthetic gel additive)) Run in TAE at 100 mv for 2 hrs with the gel rig packed in ice. (Synergel seems to make my bands a little clearer which is imperative for trying to detect 12 nt differences). I will also probably sequence these fragments - but we really would like to also ahve this visual image of the differences as well.

Any helpful hints or ideas would be very welcome. I am so glad I found this site....

Here's Hoping to some great ideas....

Dawn

no experience there.

have you tried acrylamide gels?

Hi Dawn,

Acrylamide gel is the way to go, but I still believe agarose gel should be able to show a difference of 12 bp among +600 bp PCR product.

Paulina