Feb 25, 2025

Public workspacePreparation of indexing primer stock racks V.1

DOI
dx.doi.org/10.17504/protocols.io.81wgbzod3gpk/v1
  • 1Max Planck Institute for Evolutionary Anthropology
  • MPI EVA Ancient DNA Core Unit
Ancient DNA Core Unit
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DOI: dx.doi.org/10.17504/protocols.io.81wgbzod3gpk/v1
Document Citation: Sarah Nagel, Matthias Meyer 2025. Preparation of indexing primer stock racks. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbzod3gpk/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: August 28, 2024
Last Modified: February 25, 2025
Document Integer ID: 106581
Funders Acknowledgements:
Max Planck Society
Grant ID: -
Abstract
Protocol for the preparation of tube racks in 96 SBS format containing indexing primers at a concentration of 100 µM. These stocks are needed by the Ancient DNA Core Unit of the MPI-EVA to create indexing primer aliquot plates for the automated set-up of indexing PCRs after library preparation.
Note

Lyophylized primers are dissolved to a concentration of 100 µM and transferred to FluidX tubes arranged in 96-well FluidX tube racks. P5 and P7 indexing primers are transferred to separate racks.

Materials

Reagents/consumablesSupplierCat. no.
Reagents
TE buffer*self -
P5 and P7 indexing primers, lyophilized †see indexing PCR protocol -
Consumables
1.0 ml external thread jacket tube FluidX/Brooks68-1003-11
Filter tip, Natur, 1250 µl, low retentionGreiner Bio-One778363
Filter tip, Natur, 10 µl, low retentionGreiner Bio-One771265
* See document in the Appendix for preparation of TE buffer.
† See indexing PCR protocol for primer sequences and order information.

Equipment

  • Tube decapper (e.g., Aperio 8-Channel Semi- Automatic Screw Top tube rack decapper, Brooks Life Sciences, cat. no. 46-6502)
  • Centrifuge for PCR plates (e.g., Eppendorf, cat. no. 5948000913)
  • Table-top micro-centrifuge for 1.5 ml tubes (e.g., Carl Roth Mini-Zentrifuge ROTILABO, cat. no. T464.1)

Protocol

1. Dissolve each indexing primer in TE buffer to obtain a primer stock concentration of 100 µM. Vortex thoroughly and let stand for at least 10 minutes at room temperature.

2. Vortex the primer stock tubes again and briefly spin down.

3. To prevent liquid splashing caused by electrostatic effects, first transfer 5 µl of TE buffer to the bottom of each 1 ml FluidX tube in a FluidX tube rack. Then, add 300 µl of the 100 µM primer stock solution to each tube.

Note
[Note]
Arrange the P5 indexing primer FluidX stock tubes column-wise in a 96-well FluidX tube rack. Use the following setup:
  • rack #1: P5 primers 001-096
  • rack #2: P5 primers 097-192
  • rack #3: P5 primers 193-288
  • rack #4: P5 primers 289-384
Use the same set up for the P7 indexing primer FluidX stock tubes.

4. Scan each FluidX stock tube rack using the FluidX barcode reader and save the file here:
P:\AncientDNA\indices\8bp_indices_in_FluidX

5. Store the racks at -20°C.

Appendix
Document
TE buffer
NAME
TE buffer
CREATED BY
Anna Schmidt