Author: Vikas K, Pramod Magar Sundeep Poonia and V.K. Singh
Source: Escherichia Genomics P.Ltd
Date Added: Thu Jun 27 2013
Date Modified: Sun Jun 30 2013
Abstract: Quick method to Isolate the Genomic DNA from Insect
Lysis buffer: 100 mM Tris HCL. (pH-8.0) 50 mM EDTA.Na2. (pH-8.0)
1.5 M NaCl 2% CTAB and 0.1% BME just before use
Protein Precipitation Buffer: Saturated Phenol, Phenol Chloroform
Isoamyl Alcohol (25:24:1) or Chloroform Isoamyl Alcohol (24:1)
DNA Precipitation Buffer: Absolute Isopropanol
Desalting Solution: 70% Ethanol
Storage Buffer: TE Buffer
Wash the sample in tap water followed by sterile water and dry on
Grind the sample with the help of mortar and pestle in Liquid
Nitrogen or chilled buffer manually.
Add hot saturated Phenol and vertex for 30 second at full speed.
Add 500 ul Lysis buffer (100 mM Tris HCL. (pH-8.0) 50 mM
EDTA.Na2. (pH-8.0) 1.5 M NaCl 2% CTAB and 0.1% BME (Just before use)
Incubate for 30 to 60 minutes or more at 65 degree
centigrade with occasional mixing at 10 minutes intervals
Add Phenol Chloroform Isoamyl Alcohol (25:24:1) or
Chloroform Isoamyl Alcohol (24:1) and mix thoroughly for 5 to 10
Centrifuge at 15000 rpm or 10 rpm for 10 to 15 minutes.
Transfer Supernatant in new tube.
Add equal volume pre chilled absolute Isopropanol and mix for 5
minutes and centrifuge at 10000 rpm for 10 minutes.
Discard Supernatant by saving pallets
Add equal volume 70% ethanol and nix for 5 minutes and centrifuge
at 10000 rpm for 10 minutes.
Discard supernatants and invert the tube on paper towel to drain
out excess of ethanol.
Dry the tube at room temperature for 30 minutes.
Add Tris EDTA buffer (TE buffer) to spin tube and leave for 1
hours at temperature to dissolve DNA In TE buffer
Transfer DNA in new tube and store in freeze for downstream
DNA is sufficient to perform 50 PCR reactions.
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