RFLP for 16S rRNA - (May/02/2006 )
Hi there to all
Im working on Antarctic Bacteria. My project is to derieve a phylogenetic tree from microbial diversity of soil and sediment samples,. im facing some problems in my rflp that i have to do before sending in for sequencing. Currently im using [i]Hinf1, Ncl 1 and also Rsa 1. But this enzymes dont seem to give me good cuts. Can any suggest any other RE's for 16S rRNA gene.
Thank you loads,
Basically you can use any RE. I would suggest using just grabbing a few REs at random from the freezer and try them - 10 should be enough to find a couple that work well.
if you have a sequence (evenif from related species) you can use several programs for finding appropriate enzymes, which produce sufficent numbers of fragments with suffcient size. Software: Vector NTi (now for free from invirogen), Bioedit etc. Its fatster & cheaper than to try for it in the lab.
thanks for your suggestion. Actuallly im trrying out Hae III n Hind II. Im kind of using 6 RE for time being. Send in 8 sequences n to my pleasent suprise all were different bacteria's. Im so happy. Thanks again.