Chromosome DNA Isolation from Bacteria
1. Spin down 50-100 ml well-grown bacteria, 3600 rpm,15min.
2. Resuspend bacteria with 20 ml Buffer S, immediately add 100 µl Proteinase K (10 mg/ml). Vortex to make sure no chunks.
3. Add 2 ml of 20% SDS, mix gently by inverting.
4. Incubate the mixture at 65 oC for 1 hr with inverting every 15 min.
5. Add 10 ml of phenol and 10 ml of chloroform, mix thoroughly by inverting for 5 min, spin at 3600 rpm for 20 min.
6. Transfer supernatant to a new tube, add 0.6 volume of isopropanol. Mix gently by inverting. You will see cotton-like genomic DNA.
7. Hook out the cotton-like DNA to a 1.5 ml tube; wash with cold 70% ethanol.
8. Dry DNA at RT, dissolve DNA in 500 µl H2O (50 oC or 4 oC overnight).
9. Add 5 µl DNase-free RNase A (20 mg/ml stock) to the DNA. Incubate at 37 oC for 30 min.
10. Add 500 µl phenol, mix, spin at 3600 rpm for 20 min. Repeat phenol extraction once if necessary.
11. Transfer supernatant to new tube, add 1/10 volume of 3M sodium acetate and 2 volume of ethanol, mix gently. You will see cotton-like DNA.
12. Hook out the cotton –like DNA, wash with cold 70% ethanol.
13. Dissolve DNA in 400 µl sterile H2O.
Yield: ~350 µg of genomic DNA from 100 ml of Desulfovibri vulgaris Hildenborough culture.
Solutions
Buffer S
Stock 200 ml
Tris-HCl (pH8.0) 100 mM 1M 20 ml
EDTA (pH8.0) 100 mM 0.5M 40 ml
NaCl 1.5M 5M 60 ml
CTAB 1% 10% 20 ml
CTAB/NaCl solution: 10% CTAB in 0.7 M NaCl
Dissolve 4.1 g NaCl in 80 ml distilled H2O and slowly add 10g CTAB (Sigma M-7635) while heating and stirring. Adjust final volume to 100 ml.
CTAB: Hexadecyltrimethylammonium bromide; Cetyltrimethylammonium bromide; Cetrimonium Bromide; Cetab; Centimide
Proteinase K (10 mg/ml)
Buffer
Glycerol 5 ml
1 M Tris-HCl (pH8.0) 0.1 ml
CaCl2•2H2O 0.0384 g if CaCl2, 0.029 g
H2O to 10 ml
Dissolve 100 mg Proteinase K in 10 ml of Buffer. Aliquot and store at -20oC.
3M sodium acetate (pH5.2)
Dissolve 204.15 g of sodium acetate•3H2O in 300 ml of H2O. Adjust pH to 5.2 with glacial acetic acid. Adjust final volume to 500 ml. Sterilize by autoclaving.
ที่มา http://ieg.ou.edu/protocol/Protocols/Chromosome%20DNA%20Isolation%20from%20Bacteria_48.doc
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