Dec 16, 2022

Public workspaceCost-efficient Yeast genome Flongle library 

DOI
dx.doi.org/10.17504/protocols.io.e6nvwjb2wlmk/v1
  • 1The University of Tokyo
Yutaro Hori
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DOI: 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
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 Temperature20 °C for Duration00:05:00 , then Temperature65 °C for Duration00:05:00 .



10m
Add Amount50 µL of Ampure beads (or equivalent beads) and mix well.
Wash with 75 % EtOH twice and elute with Amount11 µL of Concentration10 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 Amount1 µL of the solution to check the concentration with Qubit. The concentration needs to be at least Concentration30 ng/uL .
Assemble the following components for ligation:

AB
DNA10 uL
TLB4 uL
Quick ligase1.5 uL
AMX0.8 uL
Incubate at TemperatureRoom temperature for Duration00:30:00 .
Add Amount2.3 µL of Concentration5 Molarity (M) NaCl and incubate at TemperatureRoom temperature for Duration00:30:00 and then cfg at max speed for Duration00:20:00 .
Remove sup and add PEG wash buffer, cfg at max speed for Duration00:01:00 .
Remove sup and add Amount11 µL of 10mM Tris-HCl (pH 8.0) and incubate at Temperature4 °C forDurationOvernight .
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.