Hi Niti,

I don't have experience with metagenomic techniques but i have done a lot of work with 16S rRNA gene clone libraries from sludge samples (secondary and tertiary activated sludge). I could ask a colleague who is doing something similar. If there was one technique i avoided like the plague..and still do, it was restriction enzyme digests. However, I am not sure in what context you need them for a metagenomic library - if it is screening then there are more accurate and faster methods. Sorry, sems strange that you haven't had a reply........

Best Wishes.

..yes i was expecting a prompt discussion by researchers in here, but neverthless, to answer u ....i would say, i need restriction enzymes to digest the vector and the insert before ligation and followed by transformation in to expression vectors, i need a library to screen functionally active oxygenases.

Hi Niti,

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3235511/

I think this will link to an article which provides an earlier 'fundamental' ref'ce (Rondon et al., 2000 AEM) to BAC vector metagenomic libraries for detection of enzyme activity. I'll assume you have already looked through these. Never know!!!! Maybe post your query on linkedin also?

Good Luck.

hey, David Hayes .........thanks a lot for ur inputs..........yes i have read the article above...but why would you avoid restric. ?..and are u talking abt plate assays when u say faster screening methods?

Hi Niti,

if you are working with environmental samples then topo-TA clonning is most effective and fast to screen clonning method available now a days, though i did not performed this but this is a routine method followed in my lab to construct a metagenomic library both from soil as well as water samples