Feb 13, 2024

Public workspacessDNA2.0: Ligation mix I V.1

DOI
https://dx.doi.org/10.17504/protocols.io.6qpvr3j6bvmk/v1
  • Matthias Meyer1,
  • Sarah Nagel1,
  • Anna Schmidt1
  • 1Max Planck Institute for Evolutionary Anthropology
  • MPI EVA Ancient DNA Core Unit
Matthias Meyer
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvr3j6bvmk/v1
Document Citation: Matthias Meyer, Sarah Nagel, Anna Schmidt 2024. ssDNA2.0: Ligation mix I. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr3j6bvmk/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: February 13, 2024
Last Modified: April 10, 2024
Document Integer ID: 95158
Keywords: stranded dna library preparation, dna library preparation, stranded dna library, dna libraries for the sequencing, stranded dna, ligation mix, first adapter ligation, dna from ancient biological remain, degraded dna, dna, protocol for the preparation, sequencing, ancient biological remain
Funders Acknowledgements:
Max Planck Society
Abstract
Protocol for the preparation of Ligation mix I for the first adapter ligation in 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 Ligation mix I 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
50% PEG-8000 (w/v)Jena BioscienceCSS-256UV
100 mM ATPThermo Fisher ScientificR0441-
Consumables
5 ml screw cap tubes (rack 2d Lp W/barcode)VWRNUNC374320-BR-
* Decontamination of reagents and consumables should be performed as detailed in the documents below.


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 a 5 ml tube with Ligation mix I by adding the following reagents. Pre-mix reagents by slowly inverting the tube at least two times and spin down (full mixing is performed later, after enzyme addition).

ReagentVolume (µl)Final concentration in reaction
Water116
50% PEG-8000 (w/v)371220%
100 mM ATP46.40.5 mM
sum3874.4


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 Ligation mix I. 




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