Mar 25, 2024
ssDNA2.0: Adapter/splinter mix
- 1Max Planck Institute for Evolutionary Anthropology
Document Citation: Matthias Meyer, Anna Schmidt, Sarah Nagel 2024. ssDNA2.0: Adapter/splinter mix. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdnwwlmk/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 10, 2024
Last Modified: April 10, 2024
Document Integer ID: 93286
Funders Acknowledgement:
Max Planck Society
Grant ID: -
Abstract
Protocol for the preparation of decontaminated (i.e., nuclease-treated) Adapter/splinter mix for the first adapter ligation in 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 five (or multiples of five) tubes containing Adapter/splinter mix. Each tube 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 2-4 batches (10-20 mixes) at once.
Materials
| Reagent/consumable | Supplier | Catalogue number | Decontamination * | |
| Reagents | ||||
| Water, HPLC-grade | Merck | 270733 | UV | |
| T4 RNA ligase reaction buffer (10x) | New England Biolabs | B0216B-OTCS | - | |
| Klenow fragment (10 U/µl) | Thermo Fisher Scientific | EP0052 | - | |
| T4 polynucleotide kinase (PNK) (10 U/µl) | Thermo Fisher Scientific | EK0031 | - | |
| Tween-20 ¶ | Thermo Fisher Scientific | 11417160 | UV | |
| Adapter oligonucleotide TL181 † | IDT | - | - | |
| Splinter oligonucleotide TL159 ‡ | Eurogentec | - | - | |
| Consumables | ||||
| 0.2-ml PCR eight-tube strips | neoLab Migge | VB-0357 | UV | |
| 1.5-ml Safe-lock LoBind tubes | neoLab Migge | VB-0285 | UV | |
| 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 TL181 at 1µmol synthesis scale (Integrated DNA Technologies, desalted). Dissolve in TE buffer (See document in the Appendix for preparation of this buffer) at a concentration of 100 µM. Sequence: 5'-phosphate-AGATCGGAAGAAA[A][A][A][A][A][A][A]-TEG-biotin-3' ([A] denotes 2'-O-Methyl nucleotides)
‡ Order oligonucleotide TL159 at 1µmol synthesis scale (Eurogentec, desalted). Dissolve in TE buffer (See document in the Appendix for preparation of this buffer) at a concentration of 100 µM. Sequence: 5'-[A][A][A]CTTCCGATCTNNNNNNNN[A]-AmC6-3' ([A] denotes 2'-O-Methyl nucleotides)
Equipment
- 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)
- centrifuge for 5 ml tubes (e.g. MyFuge 5 Microcentrifuge, cat. no. 55C1005-E)
Protocol
1. Prepare the following reaction mix for decontamination of adapter oligonucleotide TL181 in a 1.5 ml Eppendorf Safe-lock tube, mix properly by flipping the tube with a finger and briefly spin down.
| Reagent | Volume (µl) | Final concentration in reaction | |
| Water | 210 | ||
| 100 µM TL181 splinter oligo | 70 | 20 µM | |
| T4 RNA Lig buffer (10x) | 35 | 1x | |
| Klenow fragment (10U/µl) | 17.5 | 0.5 U/µl | |
| PNK (10U/µl) | 17.5 | 0.5 U/µl | |
| sum | 350 |
Note
[Note]
The specified volumes in this table and the table below suffice for five tubes of Adapter/splinter mix. It is advisable to prepare 2-4 batches at once.
2. Prepare the following reaction mix for decontamination of splinter oligonucleotide TL159 in a 1.5 ml Eppendorf Safe-lock tube, mix properly by flipping the tube with a finger and briefly spin down.
| Reagent | Volume (µl) | Final concentration in reaction | |
| Water | 140 | ||
| 100 µM TL159 splinter oligo | 140 | 40 µM | |
| T4 RNA Lig buffer (10x) | 35 | 1x | |
| Klenow fragment (10U/µl) | 17.5 | 0.5 U/µl | |
| PNK (10U/µl) | 17.5 | 0.5 U/µl | |
| sum | 350 |
3. Distribute reaction mix from step 1 to seven wells of a PCR 8-strip tube, adding 50 µl to each well.
4. Distribute reaction mix from step 2 to seven wells of another PCR 8-strip tube, adding 50 µl to each well.
5. In a thermal cycler, incubate both PCR 8-strip tubes for 20 min at 37°C, followed by 1 min at 95°C to inactivate the enzymes.
6. Transfer the content of strip-tube 1 to strip-tube 2 (100 µl final volume each well) and mix by vortexing. Briefly spin tubes down.
7. In a thermal cycler, incubate the strip-tube at 95°C for 10 s and cool down to 10°C at a rate of 0.1°C/s.
8. Combine the content of all wells in a 5 ml screw-cap tube (final volume 700 µl).
9. Add 2,660 µl of water and 140 µl of 2% Tween-20 solution to obtain Adapter/splinter mix at a concentration of 2/4 µM in a final volume of 3.5 ml. Mix well by vortexing. Briefly spin tubes down in a 5 ml table centrifuge.
10. Distribute Adapter/splinter mix from step 9 to five 5 ml screw-cap tubes by adding 690 µl to each tube.
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 Adapter/splinter mix and the batch number of the Adapter/splinter mix.
Attention: The hybridization of the oligos suffices for the preparation of five separate Adapter/splinter mixes. A batch number is assigned to every unit of five Adapter/splinter mixes using Roman numerals (e.g. batch I, batch II, etc.).
11. 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. TL181-IV) or letters (e.g. TL159-x).
Appendix
Document
NAME
UV decontamination of materials CREATED BY
Elena Essel
Document
NAME
UV decontamination of reagents/buffersCREATED BY
Elena Essel
Document
NAME
TE bufferCREATED BY
Anna Schmidt