Intent: Gene isolation from sludge (provided by National Institute of Chemistry). The active sludge is a mixture of various microorganisms.
- 500 µL (0,2g) of sludge was added to microcentrifuge tube and centrifugated at 14,000 rpm 10 min.
- sludge pellet was lysed by adding equal volume of sterile glass beads and 500µL of extraction buffer hexadecyltrimethyl ammonium bromide (CTAB).
- suspension was then mixed by vortexing (1min).
- 500 µL of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the mixure then.
- the suspension was then mixed by vortexing for 1 min and frozen on ice for 1 min. We have repated this step three times (keeping on ice prevent cells from degradation).
- the mixture was centrifugated at 14,000 rpm for 10 min at 4°C. Supernatant was transferred to a new microcentrifuge tube.
- phenol extraction was repeated with 500 µL of phenol:chloroform:isoamylalcohol (24:1) and centrifugated at 14,000 rpm for 10 min at 4°C.
- the DNA was then precipitated with 0,1 volume of 3M NaOAc (sodium acetate) pH 5.5 and 0.6 volume of isopropanol at -20°C for 30 min.
- DNA pellet was washed with 70% ice cold ethanol. air dried and resuspended in 30 µL of TE buffer containing 0.002% RNase.
- The extracted DNA was used for following PCR amplification.
- The agarose gel electrophoresis showed the following results.
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