Jul 08, 2024

Public workspacePositive test extract (PTE) aliquots V.1

DOI
dx.doi.org/10.17504/protocols.io.n92ldm1enl5b/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.n92ldm1enl5b/v1
Document Citation: Anna Schmidt, Matthias Meyer 2024. Positive test extract (PTE) aliquots. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldm1enl5b/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: July 08, 2024
Document Integer ID: 93183
Funders Acknowledgements:
Max Planck Society
Grant ID: -
Abstract
This document describes the preparation of FluidX tubes containing positive test extract (PTE). It is part of the library preparation workflow of the Ancient DNA Core Unit of the MPI-EVA.
Note

A test extract prepared from animal or human material can be used as an additional positive control for in-depth testing of the performance of single-stranded library preparation, for example when testing new batches of oligonucleotides. This test extract is prepared in large volume in order to suffice for many experiments.

Materials

Reagent/consumableSupplierCatalogue numberDecontamination *
Reagents
EBT bufferself-UV
text extractself--
Consumables
FluidX screw-cap tubes in Fluidx 96-tube rackBrooks Life Sciences68-1003-11 -
Pipetting reservoir, 25 mlSigma AldrichZ680354-
* Decontamination of reagents and consumables should be performed as detailed in the documents below.

Equipment

  • FluidX rack barcode reader (Brooks Life Sciences, cat. no. 20-4018)
  • multichannel pipette (e.g. Thermo Scientific Finnpipette, cat. no. 4661050N)

Protocol

1. In a 50 ml Falcon tube, prepare PTE dilution by combining 12.5 ml EBT with 2.5 ml test extract. Mix properly.

Note
[Documentation]
Note the lot numbers, extract ID, date and initials written on the reagents in Labfolder (orange fields).


2. Get a new FluidX rack and use the FluidX barcode reader to read the bottom barcodes of all tubes in the rack.


Note
[Documentation]

Save the document containing the tube IDs under
"P:\AncientDNA\FluidXBarCodeReader\FluidX_Data\Stock_Buffer_FluidXTubes" and label rack file with rack ID, tube content, date and initials.


3. Carefully open the first row of the FluidX rack with the FluidX decapper. Keep the clean lids on the decapper to use them later for closing the tubes.

4. Pour required amount of PTE dilution into a fresh reservoir.

5. Pipette 30 µl of PTE dilution into each of the 8 FluidX tubes using a multichannel pipette.

6. Carefully close the FluidX tubes and open the next row. Repeat this step until all tubes of the rack are filled.

Note
[Labeling]

Label the FluidX rack with tube content (Positive Test Extract), date and initials. If more than one rack is prepared also add "rack x (consecutive number) of x (total amount of prepared racks)", e.g. "rack 3/4".


Note
[Note]
Optional: Get a new FluidX rack and repeat the whole procedure if you want to prepare another rack of PTEs. The PTE dilution in the reservoir can be re-filled and re-used.


7. Store PTE aliquots at -20 °C until used.


Appendix
Document
EBT buffer
NAME
EBT buffer
CREATED BY
Anna Schmidt

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