Feb 17, 2020
Marchantia genotyping (quick and dirty genomic DNA extraction)
- 1University of Cambridge;
- 2University of Cambridge, Open Plant;
- 3Plant Sciences, University of Cambridge, OpenPlant
- OpenPlant Project
Protocol Citation: Eftychis Frangedakis, Marta Marta Tomaselli, Marius Rebmann, Susana Sauret-Gueto 2020. Marchantia genotyping (quick and dirty genomic DNA extraction). protocols.io https://dx.doi.org/10.17504/protocols.io.bcmwiu7e
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: February 17, 2020
Last Modified: February 17, 2020
Protocol Integer ID: 33174
Abstract
This protocol allows for quick and dirty genomic DNA extraction. It can easily be used for genotyping with PCR. The quality of the genomic DNA extracted is not suitable for any other application.
It has been widely used in different plant species including Marchantia as in https://www.nature.com/articles/srep01532
Materials
MATERIALS
KOD Hot Start DNA PolymeraseMerck MilliporeSigma (Sigma-Aldrich)Catalog #71086-3
Take small pieces (3x3 mm) of thalli from individual plants and place in a 1.5 mL Eppendorf tube
(A in Figure).
Add 100 μl of genotyping buffer,: 100 mM Tris-HCl pH 9.5, 1M KCl, 10 mM EDTA (B in Figure).
Crush with an autoclaved micro-pestle (C in Figure).
Place the tube(s) at 80 °C for 10 min.
Add 380 μL of sterile water to each tube (D in Figure).
Use 5 μl aliquot of the extract as a template for PCR using preferably the KOD Hot start polymerase.
Note
We found KOD to be more reliable amplyfing fragments from a crude genomic DNA extract such as the one used here.
Check PCR products on a 1.5% (w/v) agarose gel.