Mar 22, 2024
Version 1
0.1xBWT buffer V.1
- Anna Schmidt1,
- Sarah Nagel1,
- Matthias Meyer1
- 1Max Planck Institute for Evolutionary Anthropology
- MPI EVA Ancient DNA Core Unit
Document Citation: Anna Schmidt, Sarah Nagel, Matthias Meyer 2024. 0.1xBWT buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdnn7lmk/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: 93162
Keywords: stranded dna library preparation, dna library preparation, stranded dna library, dna libraries for the sequencing, dna from ancient biological remain, wash buffer, degraded dna, dna, protocol for the preparation, buffer, ancient biological remain
Funders Acknowledgements:
Max Planck Society
Grant ID: -
Abstract
Protocol for the preparation of 0.1x BWT buffer (Wash buffer II) 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
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 | |
| 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 | 483.75 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% | |
| 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 ~84 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 one year from preparation.
Note
[Documentation]
Note the lot numbers, date and initials written on the reagents used for buffer preparation in Labfolder (orange fields).
Appendix