fuzzy pcr

[Beth Wapelhorst]

"PBS::NELSONJ"@PBS.DFO.CA writes:



>Hi,
>	I am doing microsatellite amplification from genomic dna and I find
>for one of the microsatellites I'm amplifying the bands (around 100-200 bp)
>always seem to be fuzzy on a 3% nusieve/1% BRL gel.  I don't think it is the gel
>as the marker looks ok and other microsatelites amplified of approx this size
>amplify up as more distinct bands.
>	Any ideas out there on this?  
>John         nelsonj at pbs.dfo.ca


Yer gettin' a lot of slippage in yer PCR. Check out the sequence - Is 
there like a string of nucleotides like ...AAAAAAAAAAA... between the 
primers? This makes for fuzzy bands. Just move the one of the primers if 
you can. Or, you could try to make the PCR more stringent, but I bet yer 
gonna have to change a primer. Also, I'd take acrylamide over agarose for 
microsatellites any day of the week.

sugar beth
weazel at seanet.com