This is a very fast mini-prep protocol which is suitable for sequence analysis and restriction digests. Although the yield is higher than Protocol D.1, there is considerable chromosomal DNA, RNA and protein contamination. Solutions Sucrose/Tris 25% sucrose 25 g sucrose 50 mM Tris pH 8.0 5 ml 1M Tris pH 8.0 up to 100 ml with Q store at room temperature Triton Lysing Mix 5% Triton X-100 5 ml Triton X-100 5% sucrose 5 g sucrose 50 mM Tris pH 7.5 5 ml 1M Tris pH 7.5 50 mM EDTA 10 ml 0.5 M EDTA pH 8.0 up to 100 ml with Q store at room temperature 2.5 M KOAc 2.5 M postassium acetate 24.5 g potassium acetate pH to 4.8 with glaicial acetic acid up to 100 ml with Q store at room temperature Procedure • Pellet 1.5 ml of an overnight culture in an eppendorf tube by spinning at14K. Resuspend in 15 ml Sucrose/Tris. Prepare Lysozyme Mixture (10mg/ml (Sigma #L6876) in Sucrose/Tris) Prepare boiling water bath • Add 100 ml Triton Lysing Mix and briefly vortex on half speed. Add 10 ml Lysozyme Mixture, invert, and incubate at room temperature for 5'. • Boil for 4' and spin at 14K for 15' in the cold room. • Remove the pellet with a toothpick and add 12 ml 2.5 M KOAc and 100 ml isopropanol; incubate at -80o for 10'. • Spin 10' and wash the pellet with 80% EtOH. • Dry and resuspend in 30 ml Q. I use 5 ml for a standard digest (RNaseA is required), and 13.5 ml for sequencing (Protocol S.1). |
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