Freeze & Squeeze: DNA from Low-melt

[BACKD at QUCDN.QueensU.CA]

Freeze squeeze protocol for extraction of DNA or RNA from agarose gels.
Ref: Locker,J. (1979) Anal. Biochem. 98; 358-367
     Tautz, D. & Renz, M. (1983) Anal. Biochem. 132; 14-19.
1)  Excise band from gel and place into 0.5ml microcentrifuge tube plugged
    at the bottom with siliconized glass wool.
2)  Add 400ul of 0.4M Tris-HCl, pH 7.8, 0.3M MaCl, 2mM EDTA and freeze
    the tube in liquid Nitrogen (dry ice also works)
3)  Pierce the bottom with a 25 gauge needle heated in a flame.
4)  Place the 0.5ml centrifuge tube (still frozen) into a 1.5ml tube
    and centrifuge at full speed for 5 minutes. If some agarose pieces
    remain, repeat the centifugation.
5)  Extract the pooled eluate with sec-butanol to reduce volume and remove
    EtBr then EtOH ppt.
6)  DNA ready for use.
RNA extraction
1)  Run a methyl mercury gel and remove the lane of interest, but don't stain
Place agarose in 10mM NaPO4, pH 7.6, 10mM B-EtSH
2)  Remove and stain a lane of markers and use to estimate position of the
    RNA band you want to extract.
3)  Remove band of interest and extract RNA as described for DNA
4)  Adjust salt and ethanol ppt RNA directly following the extraction without
    butanol extraction
5)  The RNA can be used for cDNA synthesis or translation

Don Back
Dept of Biochem
Queen's University
Kingston, Ontario