Yeast DNA purification
We will extract DNA from yeast using the MasterPure Yeast DNA Purification kit. It enables
efficient, high-yield purification of high-molecular-weight DNA from yeast and other fungi. The
protocol involves nonenzymatic cell lysis at 65°C, followed by removal of protein by
precipitation, and nucleic acid precipitation and resuspension.
Procedure
1. Scrape a single yeast colony or yeast mass (2mm in diameter) from an agar plate and
transfer to a microcentrifuge tube containing 300 l of Yeast Cell Lysis Solution
(provided).
2. Suspend the cells by pipetting the cells repeatedly using a P1000 set to 150 L.
3. Incubate the suspended cells at 65C for 15 minutes.
4. Place the sample on ice for 5 minutes. Add 150 L of MPC Protein Precipitation reagent
and mix by pipetting up and down for 10 seconds.
5. Pellet cellular debris by centrifugation in a microcentrifuge for 10 minutes at 13000 rpm.
6. Transfer the supernatant to a clean microcentrifuge tube and add 500 L of isoproponal.
Mix thoroughly by inversion.
7. Pellet the DNA by centrifugation in a microcentrifuge for 10 minutes at 13000 rpm.
8. Remove the supernatant by pipetting and discard it.
9. Wash the pellet containing the DNA with 500 L of 70% ethanol.
10. Centrifuge briefly for 1 minute at 13000 rpm. Carefully remove the ethanol by pipetting
and discard it. Remove all traces of ethanol.
11. Suspend the DNA pellet in 35 L of sterile water. Use this as your template Yeast DNA
in your PCR reactions.
Genetics
Fall 2013