May 17, 2022

Public workspaceNanopore (without barcode)

This protocol is a draft, published without a DOI.
  • 1Kaohsiung Medical College
Hsin-Mao Wu
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Protocol Citation: Hsin-Mao Wu 2022. Nanopore (without barcode). protocols.io https://protocols.io/view/nanopore-without-barcode-b9a2r2ge
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: In development
We are still developing and optimizing this protocol
Created: May 13, 2022
Last Modified: September 13, 2023
Protocol Integer ID: 62522
Abstract
Nanopore
Guidelines
1. 含DNA禁止pipetting, vortex
2. 含DNA用輕彈、輕輕spin-down
3.使用filter tip
4. 多個樣本請放在水域槽或保冷孔盤
Before start
1. 上機前先用QBIT測量DNA含量
2. 多樣本用Nanopore (with barcode) protocol
3. 依樣本數數據量決定是否加barcode
Step 1 End-prep.
Step 1 End-prep.
上NEBiocalculator計算DNA用量
並取出計算好用量的DNA
Note
50μl 中含有100fmol DNA之DDW

ds:mass⇆moles
moles→mass
輸入DNA片段長度及DNA fmol
輸出DNA mass(ng)
mass/conc.=DNA用量(μl)
加入DDW至Amount50 µL

依序加入Amount7 µL End Repair & A-Tailing Buffer, Amount3 µL End Repair & A-Tailing Enzyme Mix
加完後vortex、離心數秒
放進水浴槽：
Equipment
Veriti 96-Well Thermal Cycler
NAME
Applied Biosystems
BRAND
4375786
SKU
https://www.thermofisher.com/order/catalog/product/4375786#/4375786
LINK

[nanopore_step1]
sample要均分2管(每管30μl) 放在機器裡正中間的槽
[edit]確認流程
[run]之前改sample量
1h
Step 2 Adapter Ligation
Step 2 Adapter Ligation
取Step 1之Amount60 µL 產物
依序加入Amount5 µL AMX, Amount5 µL DI Water, Amount30 µL Ligation Buffer, Amount10 µL DNA Ligase

Ligation Buffer 輕彈至完全混合
DNA Ligase先不要拿
放進水浴槽：
Equipment
Veriti 96-Well Thermal Cycler
NAME
Applied Biosystems
BRAND
4375786
SKU
https://www.thermofisher.com/order/catalog/product/4375786#/4375786
LINK

[Nanopore_step2]
sample均分2管(每管55μl)
[edit]確認流程
[run]之前改sample量
20m
Step 3 DNA library clean-up
Step 3 DNA library clean-up
5m
5m
取Step 2之Amount110 µL 產物

加入Amount44 µL AMPure XP Beads
Note
在9F 4度冰箱使用前先搖均勻


靜置Duration00:05:00

5m
離心數秒
放置於磁座上，等待磁鐵吸附Beads
移除上清液
Note
從液面開始吸，以免吸到beads；要吸乾淨，先用大的pipette再用小的

加入Amount250 µL LFB

持續輕彈離心管Duration00:03:00
Note
小心不要噴到頂部


3m
放在磁座上 等待磁鐵吸附beads
移除上清液，7~9步驟再重複一次
Note
Ｗash兩次；Wash掉未帶有adapter再純化一次

加入Amount15 µL Elution Buffer

輕彈將磁珠彈離管壁
離心數秒；放置磁座上
Note
放較上面一些，因sample量較少
等待磁鐵吸附beads
靜置Duration00:10:00 至TemperatureRoom temperature

10m
取Amount1 µL 測QBIT

Step 4 Priming and loading
Step 4 Priming and loading