DNA extraction without phenol extraction and centrifugation.
1. Lysis: the lysis buffer (usually 0.5ml) is added to the tissue or cell ( for a 75cm2 flask, add 5ml directly to the cell). Digestion is complete within several hours at 37C (cell, 2-3h) or 55C (tissue) with agitation.
100mM Tris Hcl pH 8.5 5ml
0.5M EDTA 0.5ml
10% SDS 1ml
5M NaCl 2ml
20mg/ml Proteinase K 0.25ml
Bring up to 50ml with dd water
2. isopropanol precipitation: one volume of isopropanol is added to the lysate and the samples are mixed or swirled until precipitation is complete ( about 10-20 min ) (viscosity completely gone).
3. Recovery of precipitate: the DNA is recovered by lifting the aggregated precipitate from the solution using a disposable yellow tip. Excess liquid is dabbed off and the DNA is dispersed in a prelabeled Eppendorf tube containing, depending on the size of the precipitate, 20 to 500ul to 10mM Tris HCl, 0.1mM EDTA, pH 7.5. complete dissolution of the DNA may requires several hours of agitation at 37C or 55C (may need overnight). It is important that the DNA is completely dissolved to ensure the reproducible removal of aliquots for analysis.
Adopted from Laird PW, Nucleic Acids Research 1991,19:4293