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DNA Isolation from Insect Cells

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

Reagents Procedures
  1. Wash the sample in tap water followed by sterile water and dry on paper towel.
  2. Grind the sample with the help of mortar and pestle in Liquid Nitrogen or chilled buffer manually.
  3. Add hot saturated Phenol and vertex for 30 second at full speed.
  4. 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)
  5. Incubate for 30 to 60 minutes or more at 65 degree centigrade  with occasional mixing at 10 minutes intervals
  6. Add  Phenol Chloroform Isoamyl Alcohol (25:24:1) or Chloroform Isoamyl Alcohol (24:1) and mix thoroughly for 5 to 10 minutes
  7. Centrifuge at 15000 rpm or 10 rpm for 10 to 15 minutes.
  8. Transfer Supernatant in new tube.
  9. Add equal volume pre chilled absolute Isopropanol and mix for 5 minutes and centrifuge at 10000 rpm for 10 minutes.
  10. Discard Supernatant by saving pallets
  11. Add equal volume 70% ethanol and nix for 5 minutes and centrifuge at 10000 rpm for 10 minutes.
  12. Discard supernatants and invert the tube on paper towel to drain out excess of ethanol.
  13. Dry the tube at room temperature for 30 minutes.
  14. Add Tris EDTA buffer (TE buffer) to spin tube and leave for 1 hours at  temperature to dissolve DNA In TE buffer
  15. Transfer DNA in new tube and store in freeze for downstream processing.
  16. DNA is sufficient  to perform 50 PCR reactions.
References:
  1. Lagisz, M, Port, G.  and Wolff, K. (2010) A cost-effective, simple and high-throughput method for DNA extraction from insects. Insect Science 00, 16.
  2. Venu M. Margam, Emma W. Gachomo, John H. Shukle, Oluwole O. Ariyo, Manfredo J. Seufferheld,  and Simeon O. Kotchoni (2010) A simplified arthropod genomic-DNA extraction protocol for polymerase chain reaction (PCR)-based specimen identification through barcoding. Mol Biol Rep (2010) 37:36313635.
  3. Kamala Jayanthi PD, Rajinikanth R, Sangeetha P, Ravishankar KV, Arthikirubha A, Devi Thangam S and Abraham Verghese (2012) Rapid isolation of high molecular weight DNA from single dry preserved adult beetle of Cryptolaemus montrouzieri for polymerase chain reaction (PCR) amplification. African Journal of Biotechnology Vol. 11(90), pp. 15654-15657.
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