16S rDNA-RFLP - Non-specific banding (Nov/27/2004 )

Hello,

i do investigation on microbial diversity, where, i used to amplify 16S rRNA gene which is of 1.5 kb using specific primers. To further proceed with DNA sequencing we have funding problem.
so, i have decided to see the diversity of microbes by restriction analysis of 16S rRNA gene.
but, i had a problem with the restriction analysis, where i get small 70 to 150 bp non specific bands (faint):::; along with the major banding pattern which corresponds (only major fragments corresponds for 1.5 kb) for 1.5 kb (that is my template 16S rDNA PCR product).
i had a doubt whether it's because of incomplete digestion and repeated the experiment with prolonged incubation. but, of no use.

so kindly suggest me how to proceed,

Thanks for your suggesions in advance

ravi,
INDIA

Hi Ravi,
Did you do a pilot study in which you direct-sequence two or three samples to check whether you are using a right RE?

maybe you can visit this site. It's worth to take a look.

http://www.fermentas.com/techinfo/re/restrdigpcr.htm

Cheers,
Frank