Mar 07, 2024

Public workspacessDNA2.0: Dephosphorylation mix

DOI
dx.doi.org/10.17504/protocols.io.8epv5x5png1b/v1
  • 1Max Planck Institute for Evolutionary Anthropology
Anya Patova
Open access
DOI: dx.doi.org/10.17504/protocols.io.8epv5x5png1b/v1
Document Citation: Sarah Nagel, Anna Schmidt, Matthias Meyer 2024. ssDNA2.0: Dephosphorylation mix. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x5png1b/v1
License: This is an open access document 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
Created: January 09, 2024
Last Modified: April 10, 2024
Document Integer ID: 93177
Funders Acknowledgement:
Max Planck Society
Grant ID: -
Abstract
Protocol for the preparation of Dephosphorylation mix for automated single-stranded DNA library preparation using the ssDNA2.0 method (Gansauge et al. 2020).

References
Gansauge, M.-T., Aximu-Petri, A., Nagel, S., & Meyer, M. (2020). Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA. Nature Protocols, 15, 2279-2300.
Note

The volume of Dephosphorylation mix suffices for one 96-well library preparation plate (96 + 20 reactions to account for dead volumes and loss of reagent). It is advisable to prepare 10-20 mixes at once.

Materials
Reagent/consumableSupplierCatalogue numberDecontamination *
Reagents
Water, HPLC-grade Sigma Aldrich/Merck1153332500UV
10x T4 RNA Lig bufferNew England BiolabsB0216B-OTCS-
Tween-20 †Thermo Fisher Scientific11417160UV
Consumables
5 ml screw cap tubes (rack 2d Lp W/barcode)VWRNUNC374320-BR-
* Decontamination of reagents should be performed as detailed in the document in the Appendix.
† Use to prepare a 2% (vol/vol) solution in water. NOTE: Tween-20 is highly viscous, pipette slowly and with care.
Equipment

  • Label printer (e.g. Brady M611, cat. no. M611-EU-LABS) and tube labels (e.g. Labels for TLS2200/TLS PC Link/Polyester, cat. no. PTL-82-499)

Protocol

1. Prepare Dephosphorylation mix in a 5 ml screw-cap tube by combining the following reagents. Mix thoroughly by vortexing. Spin tube briefly in a microcentrifuge.

ReagentVolume (µl)Final concentration in reaction
Water588.8
T4 RNA Lig buffer (10x)10241x
2% (v/v) Tween-202560.05%
sum1868.8

Note
[Labeling]
Prepare tube labels using Brady printer including name of the mix, date (dd.mm.yyyy) and the name of the person who prepared the Dephosphorylation Mix.


2. Freeze at -20 °C until used.

Note
[Documentation]

Note the lot/batch numbers of the reagents used for master mix preparation in Labfolder (orange fields).

Appendix
Document
UV decontamination of reagents/buffers
NAME
UV decontamination of reagents/buffers
CREATED BY
Elena Essel