I am looking for a good vector for the cloning of gut-micflora metagenome. The source of metagenome is insect gut. I isolated a number of cultures and did 16sRna sequencing. On the other side we are doing metagenomic approach to it. I tried with PGEMT vector and the ligation reaction faliured for it. Anyone suggest the reason for ligation failure or any other good suitable vector.
Hi Subash !! what is your plan or objective !? 16sRNA sequence will give you species specification..so you can Identify the species easly with hypervariable regions...if you can do the anaylsis... check this paper..
http://www.ncbi.nlm.nih.gov/pubmed/21544196
http://lucigen.com/docs/posters/Random-Shear-Shuttle-BAC-4th-Annual-Argonne-Metagenomic-Meeting-2012-09-28.pdf
may be helpful for you
Hey mohan,
I am looking for two sets of microflora in insect gut.one is culture dependent and another one is culture independent,16sRNA useful for dependent cultures, which was completed.In another approach (culture independent technique), I am trying to find number of cultures. I tried with PGEMT vector which met failure.So now I am screening vectors.
Usually TA vector ... works good for culture dependent 16S rRNA gene sequencing as well as direct 16S rRNA gene library cloning as well....Basically most of the PCR fragments which amplified using Taq DNA polymerase preferentially adds an adenine to the 3' end of the product...this overhang is used to ligate with linerised TA vector having T overhang with form a compatibility.
Your ligation failure might be due to,
1) excess or very less of PCR product as insert
2) Impurity of salt or other ligase inhibitors
3) ligation conditions not provided...(Temp., or buffer's DTT is oxidized or ATP loss)
4) transformation inefficiency.
5) Antibiotic marker need to be checked.
6) Loss of ligase activity!!!!
All the best.
if you have already amplified metagenomic DNA with 16S primers then TA cloning kit should work and even pGEMT vector is supporting PCR product.
are you cloning after PCR amplification or before PCR?
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