Jun 11, 2026

REPLI-g Advanced DNA Single Cell Kit (WGA)

DOI
https://dx.doi.org/10.17504/protocols.io.kxygxy7wol8j/v1
  • 1University of Georgia;
  • 2Kennesaw State University
Alejandro De Santiago
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygxy7wol8j/v1
Protocol Citation: Alejandro De Santiago, Mirayana Marcelino Barros, Tiago Pereira, Hannah Budroe, Min Khant Han, Holly Bik 2026. REPLI-g Advanced DNA Single Cell Kit (WGA). protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxy7wol8j/v1
License: This is an open access  protocol  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
Protocol status: Working
We use this protocol and it's working
Created: June 20, 2024
Last Modified: June 11, 2026
Protocol  Integer ID: 102156
Keywords: protocol for single worm amplification, single worm amplification, qiagen, wga, bacterial cell, slight modification to lyse, repli
Funders Acknowledgements:
National Science Foundation
Grant ID: CAREER award (DEB-2144304)
National Science Foundation
Grant ID: Antarctic Program award (OPP-2132641)
Gordon and Betty Moore Foundation
Grant ID: Symbiosis in Aquatic Systems Initiative, grant #9326
University of Georgia Research Foundation
National Institute of General Medical Sciences of the National Institute of Health
Grant ID: 1T32GM142623
Abstract
QIAGEN's REPLI-g Advanced DNA Single Cell Kit (WGA) protocol for single worm amplification with slight modification to lyse bacterial cells.
Protocol materials
REPLI-g Single Cell Cryo-Protect ReagentQiagenCatalog #150370
REPLI-g Advanced DNA Single Cell KitQiagenCatalog #150365
Before Starting
Pick individual nematodes into tubes containing 4 µL REPLI-g Advanced Storage Buffer (REPLI-g Single Cell Cryo-Protect ReagentQiagenCatalog #150370 ). Pick one worm per tube for genome skimming of single specimens. Live specimens work best, followed by samples previously flash frozen. If using frozen samples, thaw samples and transfer worms into tubes containing Repli G Advanced Storage Buffer as quickly as possible. Specimens left at room temperature for too long will degrade quickly and not result in good sequence data.
When opening a new REPLI-g Advanced DNA Single Cell KitQiagenCatalog #150365 , prepare Buffer DLB (clear cap) by adding 250 µL H2O. Vortex to mix and centrifuge.

Thaw all the reagents:
  • DTT (purple cap)
  • Buffer DLB (clear cap)
  • Stop solution (red cap)
  • H20 sc (clear cap)
  • REPLI-g advanced sc Reaction Buffer (yellow cap)
  • REPLI-g sc DNA Polymerase (blue cap)

Note
The REPLI-g sc DNA Polymerase (blue cap) must thaw on ice!



Cell Lysis
Prepare Buffer D2 (for 12 reactions):
  • 3 µL of 1 Molarity (M) DTT (purple cap)
  • 33 µL of reconstituted buffer DLB (clear cap)



Add 3 µL of Buffer D2 to the PCR tube with nematode suspended in Repli G Advanced Storage Buffer.

Mix tube by vortexing and centrifuge briefly.
Incubate at 65 °C for 00:10:00
Note
65 °C works well for bacterial cell. For eukaryotic cells, incubate at room temperature.



10m
Add 3 µL of the Stop Solution (red cap). Mix by slowly pipetting up and down.

Amplification Reaction
2h 3m
Prepare the Amplification Master Mix:


Note
Add reagents in this order! Vortex and centrifuge before adding the DNA polymerase

  • 9 µL * n of H2O sc
  • 29 µL * n of REPLI-g advanced sc Reaction Buffer (yellow cap)
  • 2 µL * n of REPLI-g sc DNA Polymerase (blue cap)

Add 40 µL of the Amplification Master Mix to the tube containing the lysed worm. Mix by slowly pipetting up and down.

Incubate at 30 °C for 02:00:00 (if a heated lid is used, the temperature of the lid should be 70 °C )

2h
Inactivate the reactions by heating the samples for 00:03:00 at 65 °C

3m
Store DNA at -20 °C for long storage (a concentration of at least 100 ng/μL is recommended)