Mar 07, 2024

Public workspacessDNA2.0: Fill-in mix V.1

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gp3111vzp/v1
  • Sarah Nagel1,
  • Anna Schmidt1,
  • 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.dm6gp3111vzp/v1
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
Keywords: stranded dna library preparation, dna library preparation, stranded dna library, dna libraries for the sequencing, dna from ancient biological remain, degraded dna, dna, mix protocol for the preparation, protocol for the preparation, automated preparation, ancient biological remain, mix protocol, sequencing
Funders Acknowledgements:
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.
Troubleshooting
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/consumableSupplierCatalogue numberDecontamination *
Reagents
Water, HPLC-gradeSigma Aldrich/Merck1153332500UV
Klenow reaction buffer (10x)Thermo Fisher ScientificEP0052-
25 mM each dNTP Thermo Fisher ScientificR1121-
Tween-20 †Thermo Fisher Scientific11417160UV
Extension primer CL128 § Eurogentec--
Consumables
5 ml screw cap tubes (rack 2d Lp W/barcode)Thermo Fisher ScientificNUNC374320-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.

ReagentVolume (µl)Final concentration in reaction
Water3868.6
Klenow reaction buffer (10x)4641x
25 mM each dNTP46.4200 μM each
25 μM CL128 oligonucleotide11625 pmol/rct
2% Tween-20 (v/v)1450.05%
sum4640

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 buffer
CREATED BY
Anna Schmidt

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