Homemade Protocol - Instead of DNA-Extraction Kit
by Eduardo Daniel Souza-Canada, Labtimes 03/2007
Uses his one protocol for DNA-extraction: Eduardo Daniel Souza-Canada
It’s not always necessary to order the latest kit when planning the extraction of DNA. Eduardao Daniel Souza-Canada has developed a simple protocol for the isolation of genomic DNA from leaves.
I have tried and compared several approaches for extraction of genomic DNA from leaves, until I found a method that perfectly meets our needs.
The extraction method I have established is reliable and fast and works with leaves from wheat, tobacco, and Arabidopsis thaliana. There is no need to add cetyl trimethyl ammonium bromide (CTAB) and polyvinylpyrrolidon (PVP) to the extraction buffer. You may also forget about adding beta-mercaptoethanol, phenol or proteinkinase A. The genomic DNA extracted according to our protocol is ready for use in PCR or restriction digests.
- 100 mM Tris-HCl
- 700 mM NaCl
- 50 mM EDTA pH 8,0
- RNAse 10μg/μl
- pH value of extraction buffer: 8.0
Agarose gel of genomic DNA extracted from wheat leaves (left figure). PCR products using genomic DNA extracted from wheat leaves as template.
- Mince 60 to 100 mg of leaves in liquid nitrogen (We use a ball mill for grinding the leaves. This preserves them from being in direct contact to the nitrogen).
- Mix the crushed leaves with 1330 μl of prewarmed (65°C) extraction buffer and incubate for 15 minutes at 65°C. Invert the reaction tube from time to time.
- Add 650 μl Chloroform/Isoamylalcohol after cooling the sample to room temperature for one minute and gently shake for 5 minutes.
- Centrifuge 2 minutes (14000 rpm) at room temperature. Transfer the upper layer containing the DNA into a fresh tube. (Option: add 10 μl RNase at this step and incubate 10 minutes at 37 °C).
- Mix with 700 μl isopropanol and incubate 2 minutes at room temperature to allow the DNA to precipitate. Centrifuge for 5 to 10 minutes (14000 rpm) at room temperature.
- Wash the pellet 5 minutes in 1 ml ethanol (70%)
- Air-dry the pellet before dissolving it in 100 μl distilled water at room temperature (or at 65°C) for 10 minutes.
(Eduardo Daniel Souza-Canada, Institute of Plant Physiology, University Bayreuth, Germany)
Last Changed: 10.11.2012