Mar 07, 2024

Public workspacessDNA2.0: Klenow mix V.1

DOI
https://dx.doi.org/10.17504/protocols.io.ewov1qkbygr2/v1
  • Sarah Nagel1,
  • Anna Schmidt1,
  • Matthias Meyer1
  • 1Max Planck Institute for Evolutionary Anthropology
  • MPI EVA Ancient DNA Core Unit
Anya Patova
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DOI: https://dx.doi.org/10.17504/protocols.io.ewov1qkbygr2/v1
Document Citation: Sarah Nagel, Anna Schmidt, Matthias Meyer 2024. ssDNA2.0: Klenow mix. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1qkbygr2/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: 93169
Keywords: stranded dna library preparation, dna library preparation, stranded dna library, dna libraries for the sequencing, dna from ancient biological remain, mix protocol for the preparation, klenow mix, degraded dna, dna, protocol for the preparation, mix protocol, ancient biological remain, klenow, automated preparation
Funders Acknowledgements:
Max Planck Society
Grant ID: -
Abstract
Protocol for the preparation of Klenow 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.
Troubleshooting
Note

The volume of Klenow 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-gradeSigma Aldrich/Merck1153332500UV
Tween-20 † Thermo Fisher Scientific11417160UV
Klenow reaction bufferThermo Fisher ScientificEP0052-
Consumables
5 ml screw cab tubes (rack 2d Lp W / barcode)VWRNUNC374320-BR-
* Decontamination of reagents should be performed as detailed 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 the Klenow 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
Water945
Klenow reaction buffer (10x)1401x
2% Tween-20 (v/v)350.05%
sum1120

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 Klenow 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
NAME
UV decontamination of reagents/buffers
CREATED BY
Elena Essel