May 07, 2024
Version 2
0.1xBWT+SDS buffer V.2
- Anna Schmidt1,
- Sarah Nagel1,
- Matthias Meyer1,
- Elena Essel2
- 1Max Planck Institute for Evolutionary Anthropology;
- 2Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany
- MPI EVA Ancient DNA Core Unit
Document Citation: Anna Schmidt, Sarah Nagel, Matthias Meyer, Elena Essel 2024. 0.1xBWT+SDS buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo311zv4o/v2Version created by Ancient DNA Core Unit
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: May 07, 2024
Last Modified: May 07, 2024
Document Integer ID: 99370
Keywords: stranded dna library preparation, dna library preparation, stranded dna library, dna libraries for the sequencing, dna from ancient biological remain, degraded dna, dna, protocol for the preparation, wash buffer, ancient biological remain
Funders Acknowledgements:
Max Planck Society
Grant ID: -
Abstract
Protocol for the preparation of 0.1x BWT+SDS buffer (Bind and wash buffer I) 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/consumable | Supplier | Catalogue number | |
| Reagents | |||
| Water | Sigma Aldrich/Merck | 1153332500 | |
| 5 M NaCl | Sigma Aldrich/Merck | S5150-1L | |
| 1 M Tris-HCl, pH 8.0 | AppliChem | A4577,1000 | |
| 0.5 M EDTA, ph 8.0 | AppliChem | A4892,1000 | |
| Tween-20 | Thermo Fisher Scientific | 11417160 | |
| 20% SDS | Thermo Fisher Scientific | AM9820 | |
| Consumables | |||
| Square media bottle 500 ml | VWR | 391-0630 | |
| 50 ml serological pipet | Corning BV | 357550 | |
| 10 ml serological pipet | Corning BV | 357551 | |
| 5 ml serological pipet | Corning BV | 357543 | |
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.
| Reagent | Volume | Final concentration in reaction | |
| Water | 471.25 ml | ||
| 5 M NaCl | 10 ml | 100 mM | |
| 1 M Tris-HCl, pH 8.0 | 5 ml | 10 mM | |
| 0.5 M EDTA, ph 8.0 | 1 ml | 1 mM | |
| 100% Tween-20 | 0.25 ml | 0.05% | |
| 20% SDS solution | 12.5 ml | 0.50% | |
| sum | 500 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 ~71 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
[Documentation]
Note the lot numbers, date and initials written on the reagents used for buffer preparation in Labfolder (orange fields).
Appendix