Extracting the V4 region from a complete genome

[lorealmt]

Hello all!

I am new to the forum and I want to start off with a question!

Ok here goes, I hope this makes sense!!!

I wish to extract just the V4 region of 30 or so complete genomes. I have identified 2 sets of primers 515r/806r and Com1/Com2-ph but for some reason when I get the intersection via perl the seqs are over 1000bps!!! Any ideas!!

[lorealmt]

so what primers would I use to extract just the v4 region in complete genome bacterial

[Kumari Richa]

V4 Region illumina

Hi all,

I want to sequence v4 region of bacterial 16S from marine DNA sample. I have been trying using 515f and 806r primer sets (adapter, linker and barcodes included) but could not amplify the target region. then i tried to amplify the target using just the primers (excluding linker, adapter and barcode) which resulted in succesful amplification of the v4 region of the desired length. please suggest the thermal profile for v4 region and the concentration of reagents and template. I have followed protocol of the kit by BIOO SCIENTIFIC, nextflex 16s v4 amplicon sequencing kit.

Has anyone used 515f and 906r to amplify v4 region?

[trexbob]

I've been using the 515F-806R recently with adapters and barcodes on the primers. I have had a few samples not work, but I think it's more a problem with the DNA extraction for those particular samples. I've been using 30 cycles at 58 C anneal temp with NEB Q5 Hotstart 2x master mix. The primer concentration and rest of the parameters are by the book for the Q5 mix.