IGEM:Slovenia HS 2015/2009/Notebook/Test/2015/04/02

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2.4.2015 DNA extraction from sludge 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. prenesi sliko v pravi wiki protocols