Chromosome DNA Isolation from Bacteria
Chromosome DNA Isolation from Bacteria
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Spin down 50-100 ml well-grown bacteria, 3600 rpm,15min.
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Resuspend bacteria with 20 ml Buffer S, immediately add 100 µl Proteinase K (10 mg/ml). Vortex to make sure no chunks.
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Add 2 ml of 20% SDS, mix gently by inverting.
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Incubate the mixture at 65 oC for 1 hr with inverting every 15 min.
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Add 10 ml of phenol and 10 ml of chloroform, mix thoroughly by inverting for 5 min, spin at 3600 rpm for 20 min.
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Transfer supernatant to a new tube, add 0.6 volume of isopropanol. Mix gently by inverting. You will see cotton-like genomic DNA.
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Hook out the cotton-like DNA to a 1.5 ml tube; wash with cold 70% ethanol.
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Dry DNA at RT, dissolve DNA in 500 µl H2O (50 oC or 4 oC overnight).
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Add 5 µl DNase-free RNase A (20 mg/ml stock) to the DNA. Incubate at 37 oC for 30 min.
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Add 500 µl phenol, mix, spin at 3600 rpm for 20 min. Repeat phenol extraction once if necessary.
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Transfer supernatant to a new tube, add 1/10 volume of 3M sodium acetate and 2 volume of ethanol, mix gently. You will see cotton-like DNA.
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Hook out the cotton-like DNA, wash with cold 70% ethanol.
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Dissolve DNA in 400 µl sterile H2
Sterilization process
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.
Drosophila as a Model Organism
CTAB: Hexadecyl trimethyl ammonium bromide; Cetyl trimethyl ammonium 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.
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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 the final volume to 500 ml. Sterilize by autoclaving.