Table of Contents

Purpose & Strategy

Clone barcoded metagenomic library c203: tagmentation rxn (HE gDNA w/ Gibson homology) + barcoded plasmid backbone (w/ Gibson homology)

The goal is to make plasmid c.00203.pTwist_BsT-mStayGold_bboneBarcode-Gibson-Helongata using the barcoding and tagmentation strategy below:

PREPPING BARCODED PLASMID BACKBONE:

• 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 p00092 (needs to be resuspended in Annealing Buffer) with p00074 (already resuspended in AB)

Note that this protocol was done side-by-side with Add barcodes during tagmentation - Experimental Notes

Experimental Notes

July 12, 2024 - resuspending and annealing new primers, loading tagmentase with primers, running Tagmentation reaction with HE gDNA

Prepare barcoded library backbone

Starting this PCR with Platinum SuperFi Polymerase

• Dpn1 by adding 2uL to a 50ul reaction, post amplification.
• Mag bead clean up with 20ul beads, eluted in 30ul H2O

***** Stored in temp -20C eppendorf tube box ⇒ T1 *****