LPS Isolation (Darveau-Hancock Method)脂多糖分离-试验方案

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Deoxyribonuclease I

Ribonuclease

Protease/Pronase

METHODS:

Grow 1 litre of bacterial cell in an appropriate media (Proteose Peptone No. 2 for P. aeruginosa) until an O.D. 600 nm of 0.6 - 0.8
Harvest cells at 7,000 rpm for 15 minutes. Lyophilize. (Note: 1 litre of wet bacterial cells equivalent to 500 mg of dried bacterial cells.)
Resuspend 500 mg dried bacterial cells in 15 ml of 10 mM Tris-HCl, pH 8.0, 2 mM MgCl₂.
Add DNase (100 ug/ml) and RNase (25 ug/ml).
French press the cell suspension twice at 15,000 psi.
Sonicate for two 30s bursts at a probe intensity of 75.
Add DNase and RNase to bring their final concentrations to 200 ug/ml and 50 ug/ml respectively.
Incubate the suspension at 37^oC for 2 hours.
Add 5 ml of 0.5 M EDTA(tetra sodium salt)/10 mM Tris, pH 8.0; 2.5 ml of 20% SDS/10 mM Tris, pH 8.0 and 2.5 ml of 10 mM Tris-HCl, pH 8.0 to give a final volume of 25 ml, final pH approx. 9.5.
Vortex and centrifuge at 50,000 g for 30 minutes at 20^oC to remove peptidoglycan.
Save supernatant. Add pronase to give a final concentration of 200 ug/ml.
Incubate at 37^oC with constant shaking overnight.
Add two volumes of 0.375 M MgCl₂/95% EtOH. Mix and cool to 0^oC in -20^oC refrigerator.
After the sample had cooled to 0^oC, centrifuge at 12,000 g for 15 minutes at 0 - 4^oC.
Resuspend pellet in 25 ml of 0.1 M EDTA(tetra sodium salt), 2% SDS, 10 mM Tris-HCl, pH 8.0.
Sonicate at a probe intensity of 75 for two 30 s bursts.
Incubate the solution at 85^oC for 30 minutes. Cool to room temperature. Bring pH to 9.5 by addition of 4M NaOH.
Add pronase to give a final concentration of 25 ug/ml. Incubate at 37^oC overnight with constant shaking.
Add two volumes of 0.375 M MgCl₂/95% EtOH and cool solution to 0^oC as before.
Centrifuge at 12,000 g for 15 minutes at 0 - 4^oC.
Resuspend pellet in 15 ml of 10 mM Tris-HCl, pH 8.0. Sonicate at a probe intensity of 75 for two 30 s bursts.
Centrifuge at 1000 rpm for 5 minutes to remove insoluble Mg/EDTA complexes.
Step 22a.? For rough LPS, wash the pellet in small volume of water; recentrifuge and add the supernatant to the outer supernatant from Step. 22.
Add MgCl₂ to give a final concentration of 25mM. Centrifuge at 200,000 g (45K) for two hours.
Resuspend pellet in distilled water. Lyophilize if necessary.
Do KDO assay.

CHCl₃:MeOH (2:1) TREATMENT (optional)

Add equal volume of CHCl₃:MeOH (2:1) to sample. Vortex.
Centrifuge at 9K for 10 minutes.
Discard the bottom layer (retain white precipitate).
Repeat the extraction (step 1 - 3).
Drive reminiscent CHCl₃ and MeOH by vacuum suction or lyophilize sample.

LIPOPOLYSACCHARIDE OF SODIUM SALT

Dialyse the above CHCl₃:MeOH treated LPS with:

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LPS Isolation (Darveau-Hancock Method)脂多糖分离
点击： 作者：51protocol收集 来源： 日期：2007-03-18 本站论坛

REFERENCE: J. Bacteriol. 155: 831-838. (Darveau, Hancock method)

REAGENTS:

10mM Tris-HCl, pH 8.0
10 mM Tris-HCl, pH 8.0; 2 mM MgCl₂
0.5 M EDTA in 10 mM Tris-HCl, pH 8.0
20% SDS in 10 mM Tris-HCl, pH 8.0
0.375 M MgCl₂ in 95% EtOH
0.1 M EDTA, 2% SDS, 10 mM Tris-HCl, pH 8.0

0.5 mM Hepes, pH 7.4, 5 mM Na₂EDTA, pH 8.0. (five times)

5mM Hepes, pH 7.4, 50 mM NaCl. (two times)

Distilled water (two times)

Change dialysate every 8 hours.

Lyophilize sample.

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