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Isolation of Genomic DNA from Gram-positive Bacteria

Saito and Miura 1963 (translated by Jie)

1. Grow bacteria in 50 ml LB with antibiotic to late log phase or stationary phase, harvest cells at 40C, and centrifuge at 4000 rpm for 10 minutes.

1. Resuspend cell pellet in 5 ml of Solution I (50mM glucose, 25 mM Tris-HCl, pH8.0, 10 mM EDTA).Add lysozyme to a final concentration of 5 mg/ml, shake at 370C for 30 minutes.

1. Add 600 ul of 10% SDS and 300 ul of proteinase K (stock concentration 20 mg/ml) to the above mixture and let stand at 550C for 1 hour.

1. Extract DNA by adding 5 ml of TE-saturated phenol-chloroform mix well and spin at 4000 rpm for 10 minutes or longer to get a good separation. Transfer the aqueous to a clean tube and repeat the same extraction step two more times

1. Transfer the aqueous phase to a clean tube and add 250 ul of 5M NaCl and 10 ml of 100% ethanol to precipitate DNA. Spin at 40C for 10 min with a speed of 2000 rpm-5000 rpm

1. Dissolve DNA pellet in 1 ml of TE containing 200 ug/ml of RNase, let stand at 370C for 30 minutes.

1. Extract RNase by adding 1ml of TE-saturated phenol-chloroform, Then add 50 ul of 5 M NaCl and 2 ml of 100% ethanol to precipitate DNA , centrifuge at v 8000 rpm for 10 min.

1. Wash the DNA pellet with 70% ethanol, dissolve DNA in 500 ul of 0.1x TE buffer. Determine the DNA concentration and store DNA at – 200 C.