E. coli KC8 Minipreps This procedure describes our method for making DNA from KC8. Because it contatains genetic lesions that are complemented by a number of different markers used for yeast cloning vectors, this strain is highly useful for plasmid rescue. However, in our hands, DNA made from this strain by conventional miniprep methods is not suitable for sequencing. The procedure below gives high quality DNA that can be used in all applications we have so far tested. 1. Grow a KC8 transformant in 5 ml LB Amp (100ug/ml) or 1 X A or M9 minimal medium (Amp 100ul/ml) to an OD600 of ³ 1. The reason for growing cells in minimal is to allow use of medium that lacks the amino acid whose synthesis is directed by the plasmid you desire to prepare, whose marker complements the auxotrophic defect in KC8. Use of such medium may slightly increase copy number and DNA yield. 2. Spin down cells in a 15 ml Falcon tube (Beckmann J-6M centrifuge, 4.2 rotor, 4000 rpm/min, 10min, 4oC). 3. Discard supernatant, resuspend cells in 300ul SET buffer, and transfer to an eppendorf tube. 4. Add 55 ul lysozyme solution (hen egg white lysozyme, 5 mg/ml in SET buffer), 40 ml RNase solution (1 mg/ml in water or TE), and incubate on ice for 15 min. 5. Boil 1 min. 6. Add 125 ml Triton Mix and invert the tube several times to get an even distribution; incubate on ice for 10 min. 7. Spin in microfuge for 10 min at RT. 8. Take off supernatant (about 500 ml) and extract twice with an equal volume of 1:1 (vol/vol) phenol/chloroform. Discard phenol chloroform layer. 9. Chloroform extract once. Discard chloroform layer 10. Add an equal volume of isopropanol to the remaining liquid, vortex, and incubate at -70oC for 20 min. Spin for 10 min at 4 oC. Pour off supernatant carefully and wash DNA pellet with 1 ml 70% ethanol. Microfuge again for 1 min, pour off the ethanol and vacuum dry the DNA briefly. Resuspend the DNA in 50 ml TE, pH 8.0. SET buffer: 20 % (w/v) Sucrose, 50 mM Tris-HCl pH 8.0, 1 mM EDTA. Triton Mix: Mix 44.5 ml water with 37.5ml 0.5M EDTA pH 8.0, 15ml 1M Tris-HCl pH 8.0 and 3ml 10% (v/v) Triton X-100. Timm Jessen Russ Finley 4 December 1993 modified 31 January 1994 617 726 5956 617 726 6893 (fax) jessen@opal.mgh.harvard.edu finley@opal.mgh.harvard.edu