This method produces good quality LambdaDNA with yields of 1520 g lgt10 DNA per 450 l of supernatant processed. PLG tubes also can be used in conjunction with other LambdaDNA purification methods.(1)
- Grow a 10 ml stock of suitable plating bacteria in L-broth plus Mg2+ (1% Tryptone, 0.5% yeast extract, 0.5% NaCl, 1 mM MgSO4, pH 7.5) overnight at 37C.
- Dilute lambda phage stock to l05 pfu per 0.1 ml in SM buffer (0.1 M NaCI, 8 mM MgSO4, 50 mM Tris-Cl, pH 7.5, 0.01% Gelatin), then mix 0.1 ml of the lambda dilution with 0.1 ml plating bacteria in a sterile 13 x 100 mm culture tube and allow phage to adsorb to bacteria for 20 minutes at 37C.
- Add 2.5 ml melted top agarose (L-broth plus Mg2+ and 0.6% agarose, 4550C) to the tube, mix well and pour onto a 100 mm L-broth plus Mg2+ plate. Allow top agarose to harden and incubate at 37C for 8 hours. Plaques should be evident at this time.
- Chill plate for 15 minutes at 4C, add 3 ml lamda diluent (10 mM Tris-CI, pH 7.5, 8 mM MgSO4) to the plate and incubate overnight at 4C with gentle rocking.
- Collect phage-containing lambda diluent to a centrifuge tube and centrifuge at 4,000 x g for 10 minutes, 4C, to pellet debris.
- Transfer 450 l of the resultant supernatant to a pre-spun (1500 x g for 12 minutes) PLG 15 ml tube (Light or Heavy), add 4.5 l 1 mg/ml DNase I and 2.0 l 12.5 mg/ml RNase, mix and incubate 30 minutes at 37C.
- Add 11.5 l 20% SDS and 4.5 l 10 mg/ml Proteinase K to the sample, mix, and incubate 30 minutes at 37C.
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