Mar 22, 2024
Version 1
ssDNA2.0: Ligation mix II V.1
- Sarah Nagel1,
- Anna Schmidt1,
- Matthias Meyer1
- 1Max Planck Institute for Evolutionary Anthropology
- MPI EVA Ancient DNA Core Unit
Document Citation: Sarah Nagel, Anna Schmidt, Matthias Meyer 2024. ssDNA2.0: Ligation mix II. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq311xlk5/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: 93166
Keywords: stranded dna library preparation, dna library preparation, stranded dna library, ligation mix ii protocol for the preparation, dna libraries for the sequencing, ligation mix ii protocol, dna from ancient biological remain, degraded dna, ligation mix ii, dna, protocol for the preparation, ancient biological remain, sequencing, automated preparation
Funders Acknowledgements:
Max Planck Society
Grant ID: -
Abstract
Protocol for the preparation of Ligation mix 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 five (or multiples of five) tubes containing Ligation mix II. Each tube suffices for a 96-well library preparation plate (containing 96 + 20 reactions to account for dead volumes and loss of reagent). It is advisable to prepare 2-4 batches (10-20 tubes) at once.
Materials
| Reagent/consumable | Supplier | Catalogue number | Decontamination * | |
| Reagents | ||||
| Water, HPLC-grade | Sigma Aldrich/Merck | 11553332500 | UV | |
| 5M NaCl | Sigma Aldrich/Merck | S5150-1L | UV | |
| TE buffer ¶ | self | UV | ||
| T4 DNA ligase buffer (10x) | Thermo Fisher Scientific | EL0013 | - | |
| 50% PEG-4000 (w/v) | Jena Bioscience | CSS-253 | UV | |
| Tween-20 † | P5927-100ML | P5927-100ML | UV | |
| Adapter oligonucleotide CL53 ‡ | Sigma-Aldrich (Merck) | - | - | |
| Adapter oligonucleotide TL178 § | Eurogentec | - | - | |
| Consumables | ||||
| 1.5-ml LoBind Safe-lock tubes | neoLab Migge | VB-0285 | UV | |
| 0.2-ml PCR eight-tube strips | neoLab Migge | VB-0357 | UV | |
| 5 ml screw cap tubes (rack 2d Lp W/barcode) | VWR | NUNC374320-BR | UV | |
* Decontamination of reagents and consumables should be performed as detailed in the documents in the Appendix.
¶ See document in the Appendix for preparation of TE buffer
† Use to prepare a 2% (vol/vol) solution in water. NOTE: Tween-20 is highly viscous, pipette slowly and with care.
‡ Order oligonucleotide CL53 at 0.2 µmol synthesis scale (Sigma-Aldrich/Merck, desalted). Dissolve in TE buffer at a concentration of 500 µM. Sequence: 5'-CGACGCTCTTC-ddC-3' (ddC denotes a dideoxy cytidine)
§ Order oligonucleotide TL178 at 10 µmol synthesis scale (Eurogentec, desalted). Dissolve in TE buffer at a concentration of 500 µM. Sequence: 5'-phosphate-GGAAGAGCGTCGTGTAGGGAAAGAGTGTA-3'
Equipment
- Benchtop thermo mixer (e.g. ThermoMixer C; Eppendorf, cat. no. 5382000015)
- Thermal cycler for PCR strip tubes (e.g. Veriti 96-Well fast Thermal Cycler, cat. no. 4375305)
- 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 double-stranded adapter by combining the reagents below in a 1.5 ml Eppendorf Safe-Lock tube. Mix properly, spin down and aliquot the mix to four wells of a PCR strip-tube (75 µl each well).
| Reagent | Volume (µl) | Final concentration in reaction | |
| TE buffer | 57 µl | ||
| 5 M NaCl | 3 µl | 50 mM | |
| 500 µM CL53 | 120 µl | 200 µM | |
| 500 µM TL178 | 120 µl | 200 µM | |
| Sum | 300 µl |
Note
[Note]
The specified volumes in the table suffice for five tubes of master mix. It is advisable to prepare 2-4 batches of the double-stranded adapter at once, corresponding to the number of master mix tubes that will be prepared.
2. Incubate PCR strip-tube(s) in thermal cycler using the following profile: 95 °C 10 sec, ramp to 14 °C at 0.1°C/s.
Note
[Documentation]
Note the lot/batch numbers of the reagents used for double-stranded adapter preparation in Labfolder (orange fields).
Attention: Batches of oligonucleotides are labelled with Roman numerals (e.g. TL178 - VIII) or letters (e.g. CL53 - A).
3. Combine the contents of all four wells into a 1.5 ml Eppendorf Safe-Lock tube.
4. Add 300 µl TE buffer to obtain 600 µl of 100µM CL53/TL178. Mix by vortexing and spin down.
5. Prepare the Ligation mix II in 5 ml screw-cap tubes by combining the following reagents. Mix thoroughly by vortexing and spin tubes briefly in a microcentrifuge.
Note
[Note]
CRITICAL STEP: White precipitate may be present in the ligation buffer after thawing. Heat the buffer vial briefly in a thermo mixer to 37 °C and vortex until the precipitate has dissolved.
| Reagent | Volume (µl) | Final concentration in reaction | |
| Water | 3364 | ||
| T4 DNA ligase buffer (10x) | 464 | 1x | |
| 50% PEG-4000 (w/v) | 464 | 5% | |
| 2% Tween-20 (v/v) | 116 | 0.05% | |
| 100 µM adapter CL53/TL178 | 116 | 2.5 µM | |
| sum | 4524 |
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 Ligation mix II and the batch number of the Ligation mix II.
Attention: Since each tube of hybridized oligonucleotides suffices for the preparation of five tubes of Ligation mix II, a batch number is assigned to every unit of five Ligation mix II tubes using Roman numerals (e.g. batch I, batch II, etc.).
6. 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).
6. Freeze at -20 °C until used.
Appendix