Mar 14, 2020

Public workspaceLibrary preparation from a single amplicon pool

DOI
dx.doi.org/10.17504/protocols.io.bdm7i49n
  • 1University of Birmingham
Josh Quick
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DOI: dx.doi.org/10.17504/protocols.io.bdm7i49n
External link: http://lab.loman.net/protocols/
Protocol Citation: Josh Quick 2020. Library preparation from a single amplicon pool. protocols.io https://dx.doi.org/10.17504/protocols.io.bdm7i49n
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: March 14, 2020
Last Modified: March 14, 2020
Protocol Integer ID: 34207
Abstract
This is a subprotocol for generating a library from a single amplicon pool
Attachments
Icon for One-pot native barcoding protocol (1).pdf
One-pot native barco...
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Safety warnings
See SDS (Safety Data Sheet) for safety warnings and hazards.
Set up the following reaction for each sample:

Component Volume

DNA amplicons (5ng/ul) Amount10 µL
Nuclease-free water Amount2.5 µL
Ultra II End Prep Reaction Buffer Amount1.75 µL
Ultra II End Prep Enzyme Mix Amount0.75 µL
Total Amount15 µL

Incubate at room temperature for Duration00:05:00
Incubate at Temperature65 °C f for Duration00:05:00
Incubate on ice for Duration00:01:00

Clean-up end-repair reaction using 1x SPRI beads
Protocol
Amplicon clean-up using SPRI beads
NAME

Amplicon clean-up using SPRI beads

CREATED BY
Josh Quick

Vortex SPRI beads thoroughly to ensure they are well resuspended, the solution should be a homogenous brown colour.

ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880

Critical
Add an equal volume (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add Amount50 µL SPRI beads to a Amount50 µL reaction.

Pulse centrifuge to collect all liquid at the bottom of the tube.

Incubate for Duration00:05:00 at room temperature.

Place on magnetic rack and incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completely clear.

Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
Add Amount200 µL of room-temperature Concentration70 % volume ethanol to the pellet.



Carefully remove and discard ethanol, being careful not to touch the bead pellet.
Go togo to step #3.7 and repeat ethanol wash.

Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
With the tube lid open incubate for Duration00:01:00 or until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).

Resuspend pellet in Amount30 µL Elution Buffer (EB), mix gently by either flicking or pipetting and incubate for Duration00:02:00 .
ReagentElution Buffer (EB)QiagenCatalog #19086



Place on magnet and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.
Quantify Amount1 µL product using the Quantus Fluorometer using the ONE dsDNA assay.
ReagentQuantiFluor(R) ONE dsDNA System, 100rxnPromegaCatalog #E4871

Equipment
Quantus
NAME
Fluorometer
TYPE
Promega
BRAND
E6150
SKU
https://www.promega.co.uk/products/microplate-readers-fluorometers-luminometers/fluorometers/quantus-fluorometer
LINK


Quantify the barcoded amplicon pools using the Quantus Fluorometer using the ONE dsDNA assay.
Protocol
DNA quantification using the Quantus fluorometer
NAME

DNA quantification using the Quantus fluorometer

CREATED BY
Josh Quick

Remove Lambda DNA 400 ng/µL standard from the freezer and leave on ice to thaw. Remove ONE dsDNA dye solution from the fridge and allow to come to room temperature.

ReagentQuantiFluor(R) ONE dsDNA System, 500rxnPromegaCatalog #E4870



Set up two Amount0.5 mL tubes for the calibration and label them 'Blank' and 'Standard'

Add Amount200 µL ONE dsDNA Dye solution to each tube.
Mix the Lambda DNA standard 400 ng/µL standard by pipetting then add Amount1 µL to one of the standard tube.

Mix each sample vigorously by vortexing for Duration00:00:05 and pulse centrifuge to collect the liquid.
Allow both tubes to incubate at room temperature for Duration00:02:00 before proceeding.

Selection 'Calibrate' then 'ONE DNA' then place the blank sample in the reader then select 'Read Blank'. Now place the standard in the reader and select 'Read Std'.
Set up the required number of Amount0.5 mL tubes for the number of DNA samples to be quantified.
Note
Use only thin-wall, clear, 0.5mL PCR tubes such as Axygen #PCR-05-C


Label the tubes on the lids, avoid marking the sides of the tube as this could interfere with the sample reading.
Add Amount199 µL ONE dsDNA dye solution to each tube.

Add Amount1 µL of each user sample to the appropriate tube.
Note
Use a P2 pipette for highest accuracy.


Mix each sample vigorously by vortexing for Duration00:00:05 and pulse centrifuge to collect the liquid.

Allow all tubes to incubate at room temperature forDuration00:02:00 before proceeding.

On the Home screen of the Quantus Fluorometer, select `Protocol`, then select `ONE DNA` as the assay type.
Note
If you have already performed a calibration for the selected assay you can continue, there is no need to perform repeat calibrations when using ONE DNA pre diluted dye solution. If you want to use the previous calibration, skip to step 11. Otherwise, continue with step 9.

On the home screen navigate to 'Sample Volume' and set it to Amount1 µL then 'Units' and set it to ng/µL.

Load the first sample into the reader and close the lid. The sample concentration is automatically read when you close the lid.
Repeat step 16 until all samples have been read.
The value displayed on the screen is the dsDNA concentration in ng/µL, carefully record all results in a spreadsheet or laboratory notebook.
Set up the following AMII adapter ligation reaction:

Component Volume

End-repaired amplicon pools Amount30 µL
Ligation Buffer (LNB) Amount10 µL
Adapter Mix (AMX) Amount5 µL
Quick T4 DNA Ligase Amount5 µL
Total Amount50 µL

Note
There will be some variation in clean-up efficiencies but expect to carry around 80% through a clean-up.

Incubate at room temperature for Duration00:10:00
Add Amount50 µL (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting.
Note
Vortex SPRI beads thoroughly before use to ensure they are well resuspended, the solution should be a homogenous brown colour.


Pulse centrifuge to collect all liquid at the bottom of the tube.
Incubate for Duration00:05:00 at room temperature.

Place on magnetic rack and incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completely clear.

Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
Add Amount250 µL SFB and resuspend beads completely by pipette mixing.

Note
SFB will remove excess adapter without damaging the adapter-protein complexes. Do not use 70% ethanol as in early clean-ups.

Pulse centrifuge to collect all liquid at the bottom of the tube.
Remove supernatant and discard.
Repeat steps 14-16 to perform a second SFB wash.
Pulse centrifuge and remove any residual SFB.
Note
You do not need to allow to air dry with SFB washes.

Add Amount15 µL EB and resuspend beads by pipette mixing.

Incubate at room temperature for Duration00:02:00 .

Place on magnetic rack.
Transfer final library to a new 1.5mL Eppendorf tube.