Dec 16, 2022

Cost-efficient Yeast genome Flongle library 

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwjb2wlmk/v1
  • 1The University of Tokyo
Yutaro Hori
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwjb2wlmk/v1
Protocol Citation: Yutaro Hori 2022. Cost-efficient Yeast genome Flongle library . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjb2wlmk/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 16, 2022
Last Modified: December 16, 2022
Protocol  Integer ID: 74086
Keywords: efficient yeast genome flongle library, flongle library, genome, yeast, flongle, library
Abstract
A cost-efficient protocol for constructing a Flongle library.
Materials
PEG/NaCl precipitation buffer (see Rocky Mountain adventure protocol)

AB
PEG 80009% (w/v)
NaCl1 M
Tris-HCl (pH 8.0)10 mM

PEG wash buffer

Dilute PEG/NaCl precipitation buffer with the same amount of water.
Assemble the following components to end repair and add A to the genomic sample:

AB
Ultra II buffer1.75
End repair buffer1.75
Ultra II enzyme1
End repair enzyme1
DNAx (500 ng)
MQ44.5 - x
Incubate at 20 °C for 00:05:00 , then 65 °C for 00:05:00 .



10m
Add 50 µL of Ampure beads (or equivalent beads) and mix well.
Wash with 75 % EtOH twice and elute with 11 µL of 10 millimolar (mM) Tris-HCl (pH 8.0).
Keep the tube on the magnetic rack and do not disturb the beads while washing with 75% EtOH.
Use 1 µL of the solution to check the concentration with Qubit. The concentration needs to be at least 30 ng/uL .
Assemble the following components for ligation:

AB
DNA10 uL
TLB4 uL
Quick ligase1.5 uL
AMX0.8 uL
Incubate at Room temperature for 00:30:00 .
Add 2.3 µL of 5 Molarity (M) NaCl and incubate at Room temperature for 00:30:00 and then cfg at max speed for 00:20:00 .
Remove sup and add PEG wash buffer, cfg at max speed for 00:01:00 .
Remove sup and add 11 µL of 10mM Tris-HCl (pH 8.0) and incubate at 4 °C forOvernight .
Check the concentration with Qubit.

1h 21m
Assemble the following priming solution:

AB
FLB2.5 uL
FB97.5 uL
and the library mix solution:

AB
DNAx uL (100 ng)
SB II10 uL
LB II4 uL
MQ6 - x uL
. Load them and start sequencing.