DNA isolation: procedure, buffers.......pptx

DNA isolation

1. DNA ISOLATION

2. Procedure Grind 50 mg of leaf material to a fine powder using quartz sand using pestle and mortar. Transfer the powdered material to 700 µl of pre-warmed Extraction buffer and 700 µl of 2X CTAB buffer and incubate for 60 min at 60° C with occasional stirring. Extract with equal volume of Phenol: Chloroform: Isoamyl alcohol (25:24:1). C entrifuge at 10,000 rpm for 15 min at room temperature (20° C). Separate the aqueous phase and transfer to a fresh tube.

3. Add 2 µl Of RNase A (10 mg/ml) to final concentration of 50 mg/ml and incubate for 30 min at 37' C (optional step) . Extract with an equal volume of chloroform: isoamyl alcohol (24:1) at 10,000 rpm for 10 min. To the aqueous phase add 0.6 volumes of ice-cold isopropanol and incubate at -20°C for30-60 min. Centrifuge at 10,000 rpm for 10 min at 4° C. Wash the DNA pellet obtained with 70% ethanol and 10 mM ammonium acetate. Dry the DNA pellet and dissolve in 100 µl of water or low concentration TE buffer.

4. BuffeR preparation

5. More info Phenol:chloroform:isoamyl alcohol - to  promote the partitioning of lipids and debris into an organic phase, leaving the DNA in the aqueous phase . CTAB - cetyltrimethylammonium bromide (CTAB)  disrupts cell membranes. SDS buffer - SDS is used to solubilize membrane and used in Bacterial DNA Extraction, remove Barriers of DNA and Histones.  RNase - to  reduce contaminating RNA. Ice cold isopropanol - The cold alcohol helps the DNA precipitate (solidify and appear) more quickly. TE buffer - TE ( Tris -EDTA) buffer is widely used  as a storage or dilution buffer solution. The primary function of the Tris -EDTA buffer is to solubilize nucleic acids while protecting them from enzymatic lysis.