Multiplex pcr trouble - (Jan/09/2009 )

hi..
i'am doing my final research which is use multiplex pcr.
now, i've trouble with it..
i've to amplify 4 genes, and the problem is 2 of my genes have closer size of amplicons..
one is 309 bp and the other is 320 bp..
i don't know how to visualization it using agarose electrophoresis..
for info, i have tried agarose 2% and 3% in various voltage and time condition, but still i can't separate the 2 bands (309 bp and 320 bp), and i used 20 x 20 cm chamber..
can i separate it using agarose electrophoresis?
somebody help me!!

I used 1% of agarose gel with 100 volt and let it run for approximately 1 hour or 3 quarter of the gel. however i am measuring 1500 bp of size for bacteria. maybe the voltage should be higher and the time should be longer so that the band can separate nicely or it could be your sample is 309bp or 320 bp of size. how about the separation of the gene ladder?
i have problem that my gene ladder cannot clearly seen even we had vortex it for a long time and the concentration is not constant. we used nile blue A as our dye, we suspect it was caused by the nile blue A but normally it can be seen clearly. Is this condition caused by the gene ladder mixture of it was caused by the nile blue? im not suspecting the gel because all this while we had used 1% agarose and it works... please someone.. help me.. i need suggestion to burst my idea.. please... i have 3 months left to finish this research..

Cella,

Try using Metaphor agarose that can separate up to 5bp bands.

Nisah,
have you tried a new tube of ladder? usually dyes won't give problems but try mixing your stock dyes properly as well.

Cheers,
Chris