Table of Contents

Strategy & Purpose

After reading the Bacterioides/Boba-Seq paper, we want to do more diligence on using blunt end ligation versus tagmentation as our strategy for fragmenting genomes.

See also Designing a replicative plasmid for barcoding a fragmented genome

Preparation

• [x] 🛍️ Need to order reagents for tapestation - order both
• [x] 🛍️ Need to order phi29 kit - order small kit to test out. Alternatively use TempliPhi kit that we’ve used before. - ordered NEB kit through Fisher
• [x] 🛍️ Order Tagmentation reagents and consumables

Gibson Design

Here’s an unloaded tagmentase, and the manual that has some nice instructions in it. This may be the same as the thermo unloaded tagmentase that we ordered ( ✅TODO check). See also the METa paper.

See Figma board that describes the tagmentation reaction we’re going to be using in more detail, also shown below:

Is the polymerase gap filling a separate reaction? It probably happenins as part of the gibson reaction

Steps to follow:

• Replace the multiple cloning site in your destination vector with the following sequence: with linearization occurring at the slash (/): 5’- AGATGTGTATAAGAGACAG/CTGTCTCTTATACACATCT-3’. These are the homology sites we’ll later use for cloning with Gibson/USER.
• Order the following three oligos. The underlined portions are the “Tn5 mosaic ends”, which the tagmentase enzyme recognizes. The NNNNN’s can be anything and get removed as part of the cloning.
  • Mosaic end_reverse: **5’Phos-**CTGTCTCTTATACACATCT
  • Mosaic end_Adapter A: NNNNNNNNNNNNNNAGATGTGTATAAGAGACAG
  • Mosaic end_Adapter B: NNNNNNNNNNNNNNAGATGTGTATAAGAGACAG
• Do the tagmentation reaction
• Gel purify your desired tagmented fragment size