Mar 21, 2024

Public workspaceStringency wash buffer V.1

DOI
https://dx.doi.org/10.17504/protocols.io.kqdg3xndeg25/v1
  • Anna Schmidt1,
  • Sarah Nagel1,
  • 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.kqdg3xndeg25/v1
Document Citation: Anna Schmidt, Sarah Nagel, Matthias Meyer 2024. Stringency wash buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3xndeg25/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: 93168
Keywords: stranded dna library preparation, dna library preparation, stringency wash buffer protocol for the preparation, stranded dna library, dna libraries for the sequencing, stringency wash buffer protocol, stringency wash buffer, dna from ancient biological remain, degraded dna, dna, wash buffer, protocol for the preparation, automated preparation, sequencing, ancient biological remain
Funders Acknowledgements:
Max Planck Society
Grant ID: -
Abstract
Protocol for the preparation of Stringency wash buffer 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

This protocol describes the preparation of 500 ml buffer.

Materials
Reagent/consumableSupplierCatalogue number
Reagents
Water Sigma Aldrich/Merck1153332500
20x SSC bufferThermo Fisher ScientificAM9770
20% SDS solutionThermo Fisher ScientificAM9820
Consumables
Square media bottle 500 mlVWR391-0630
50 ml serological pipetCorning BV357550
5 ml serological pipetCorning BV357543

Equipment

  • Serological pipette controller (e.g. battery-powered pipetting aid ROTILABO, cat. no. TC16.1)

Protocol

1. Prepare the buffer in a 500 ml square media bottle by adding the following reagents. Use the glass pipette for transfer of large volumes (> 1 ml). Mix reagents by shaking the bottle.

ReagentVolumeFinal concentration in reaction
Water495 ml
20x SSC buffer2.5 ml0.1x
20% SDS solution2.5 ml0.1%
sum500 ml

Note
[Note]

It is also acceptable to use the scale of the bottle to fill up to 400 ml with water, then adding the remaining 95 ml using the glass pipette.
2. Review the protocol in which the buffer is used to determine whether the buffer should be decontaminated using UV treatment. Instructions for UV-decontamination are provided in the Appendix.

Note
[Labeling]

Label the bottle with the buffer name, batch ID, date and the initials of the person who prepared the buffer.

Attention: Every single bottle prepared at the same day gets a new batch ID. Name the batches with Roman numerals (e.g. batch I, batch II, etc.)

3. Store the buffer at room temperature until used. Shelf life is at least two months from preparation.

Note
[Note]

Note the lot numbers and the date and initials of the reagents used for the preparation in Labfolder (orange fields).



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