Smash 'n' Grab
A 10-Minute DNA Preparation from Yeast: "Smash 'n' Grab"*
- Phenol is highly corrosive and can cause severe burns
Gloves, protective clothing and safety glasses should be worn when handling it. All manipulations should be carried out in a chemical hood. Any areas of skin that come in contact with phenol should be rinsed with a large volume of water or polyethylene glycol 400 and washed with soap and water; ethanol should not be used.
- Chloroform is irritating to the skin, eyes, mucus membranes and upper respiratory tract
It should only be used in a chemical hood. Gloves and safety goggles should also be worn.
- Chloroform is a carcinogen and may damage the liver and kidneys
- Nitric acid is volatile and should be used in a hood
Concentrated acids should be handled with great care; gloves and a face protector should be worn.
Release of Plasmid for Transformation of E. coli or yeast
- Grow small cultures (at least 1.4 ml) overnight at 30°C in a medium that maintains selection for the plasmid DNA, such as SC-urea.
- Fill a 1.5-ml microfuge tube with the culture and collect the cells by a 5-second centrifugation in a microfuge.
- Decant the supernatant and briefly vortex the tube to resuspend the pellet in the residual liquid.
- Add 0.2 ml of [2% Triton X-100, 1% SDS, 100mM NaCl, 10mM Tris Cl (pH 8), 1mM Na2-EDTA]. Add 0.2 ml of [phenol:chloroform:isoamyl alcohol (25:24:1)]. Add 0.3 g of acid-washed glass beads.
- Vortex for 2 minutes.
- Centrifuge for 5 minutes in a microfuge.
- Ethanol precipitate and resuspend in 50 µl H2O or 10mM Tris.
*From Hoffman and Winston Gene 57:267-272.