Mar 07, 2024
ssDNA2.0: Fill-in mix
- 1Max Planck Institute for Evolutionary Anthropology
Document Citation: Sarah Nagel, Anna Schmidt, Matthias Meyer 2024. ssDNA2.0: Fill-in mix. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3111vzp/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: 93161
Funders Acknowledgement:
Max Planck Society
Grant ID: -
Abstract
Protocol for the preparation of Fill-in 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.
Note
The volume of Fill-in 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/consumable | Supplier | Catalogue number | Decontamination * | |
| Reagents | ||||
| Water, HPLC-grade | Sigma Aldrich/Merck | 1153332500 | UV | |
| Klenow reaction buffer (10x) | Thermo Fisher Scientific | EP0052 | - | |
| 25 mM each dNTP | Thermo Fisher Scientific | R1121 | - | |
| Tween-20 † | Thermo Fisher Scientific | 11417160 | UV | |
| Extension primer CL128 § | Eurogentec | - | - | |
| Consumables | ||||
| 5 ml screw cap tubes (rack 2d Lp W/barcode) | Thermo Fisher Scientific | NUNC374320-BR | - | |
* Decontamination of reagents and consumables 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.
§ Order oligonucleotide CL128 at 10.0 μmol synthesis scale (Eurogentec, HPLC purified). Dissolve in TE buffer (See document in the Appendix for preparation of this buffer) at a concentration of 25 µM. Sequence: 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCC*G*A*T*C*T-3' (* denotes phosphothioate (PTO) linkages).
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 Fill-in mix in a 5 ml screw-cap tube by combining the following reagents. Mix thoroughly by vortexing. Spin tube briefly in a microcentrifuge.
| Reagent | Volume (µl) | Final concentration in reaction | |
| Water | 3868.6 | ||
| Klenow reaction buffer (10x) | 464 | 1x | |
| 25 mM each dNTP | 46.4 | 200 μM each | |
| 25 μM CL128 oligonucleotide | 116 | 25 pmol/rct | |
| 2% Tween-20 (v/v) | 145 | 0.05% | |
| sum | 4640 |
Note
[Labeling]
Prepare tube labels using Brady printer including name of the mix, date (dd.mm.yyyy), the name of the person who prepared the Fill-in mix and the batch number of the oligo used.
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).
Attention: Batches of oligonucleotides are labelled with Roman numerals (e.g. CL128 - VIII).
Appendix
Document
NAME
TE bufferCREATED BY
Anna Schmidt
Document
NAME
UV decontamination of reagents/buffersCREATED BY
Elena Essel