Table of Contents

Purpose & Strategy

Basically, do exactly the same as Add barcodes during PCR (public copy) but with a different assembly step so we don’t have cloning issues, using USER cloning to generate sticky ends & ligate them together.

The problem with First First time: fragment genomes (public copy) was that the AarI restriction enzyme cut many of the genome inserts, reducing our library size and biasing against bigger inserts. One way around this is to generate the sticky ends in away that doesn’t cut the genome, like Uracil-excision:

Therefore, the overall goal is to:

Clone barcoded metagenomic library : tagmentation rxn (HE gDNA w/USER primer) + barcoded plasmid backbone (w/ USER primer)

The goal is to make plasmid using the barcoding and tagmentation strategy below:

General experimental overview:

PREPPING BARCODED PLASMID BACKBONE:

NOTE: Must use Uracil-tolerant polymerase for this step!

• Primers need to be resuspended in water (100uM)

TAGMENTATION:

• H. elongata gDNA (same aliquot as before)

• Follow whole @Tagmentation Protocol protocol

  • Need to anneal and load primers @p00105 (needs to be resuspended in Annealing Buffer) with @p00074 (already resuspended in AB)

  NOTE: Must use Uracil-tolerant polymerase for gap-filling in tagmentation!!!!!!!!!

USER CLONING:

After PCR & tagmentation, DNA is purified and mixed in the USER cloning according to kit instructions.