Preparation of Genomic DNA from Mammalian Sperm

Alexandra Weyrich1

1 Evolutionary Genetics (FG2), Leibniz Institute for Zoo and Wildlife Research (IZW), Berlin, Germany
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.13
DOI:  10.1002/0471142727.mb0213s98
Online Posting Date:  April, 2012
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Abstract

Semen consists of spermatozoa and the seminal fluid, also called seminal plasma. This fluid is important for the survival of the spermatozoa, but may decrease the purity and thus quality of the DNA due to its fructose and protein content. In this protocol, spermatozoa are washed with ethanol to remove the fluid. The spermatozoa themselves are protected by a membrane rich in disulfide bonds, which impede cell lysis and thus hamper DNA isolation. To break disulfide bonds a strong antioxidant, such as dithiothreitol (DDT), is necessary. Similar to other protocols for DNA isolation from other mammalian tissues, proteinase K and SDS are included in the lysis reaction. After lysis of the sperm cells, the DNA is precipitated using ethanol and then redissolved in TE‐buffer or ddH2O. Curr. Protoc. Mol. Biol. 98:2.13.1‐2.13.3. © 2012 by John Wiley & Sons, Inc.

Keywords: testis; nucleic acids; mammal; sperm; disulfide bonds

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • Mammalian ejaculate
  • 70% and 75% Ethanol
  • Lysis buffer (see recipe)
  • Triton‐X100
  • Dithiothreitol (DDT; prepare fresh or store in aliquots at −20°C)
  • Proteinase K
  • Glycogen
  • 3 M sodium acetate (NaAc; filter before use)
  • Absolute ethanol
  • TE buffer, pH 8 ( appendix 22)
  • 1.5‐ml tubes
  • Thermo shaker at 50°C
  • SpeedVac, optional
  • Additional reagents and equipment for measuring DNA concentration ( appendix 3D)
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Figures

Videos

Literature Cited

Literature Cited
   Gebert, C.M. 2004. Characterization of gene expression and methylation patterns of the bovine IGF2 gene in gametes and preimplantation embryos of different origins. Dissertation, University of Veterinary Medicine Hannover, Germany.
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