5 Amplifying cleaning and pooling DNA metabarcoding libraries

Author:

CALeDNA

Keywords:

DNA barcoding,metabarcoding,PCR,Environmental DNA,Next Generation Sequencing,CALeDNA

Subtitles:

we do PCR three times for each barcode region we want to sequence in most cases that's 15 PCRs we pull the triplicates and clean them with magnetic beads that bind to the DNA we want so we can wash away the other drunk [Music] next we quantify how much DNA we have and pull together the same number of copies of each barcode for each sample [Music] then we do another PCR reaction with indexing tags instead of primers this gives each sample a unique name tag so we can tell them apart after sequencing all these tagged samples get mixed into one final pool and sent on dry ice to the genome sequencing facility