Genotyping Embryonic Stem (ES) Cells

Jennifer V. Schmidt1

1 University of Illinois at Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 15.2
DOI:  10.1002/0471140856.tx1502s09
Online Posting Date:  November, 2001
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Abstract

This unit describes a means for isolating DNA from clones cultured in 96‐well plates. The extracted DNA can be used for Southern blotting or PCR to verify the genotype of the ES clone.

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of ES Cell Clones by Southern Blot Hybridization
  • Alternate Protocol 1: Analysis of ES Cell Clones by PCR
  • Support Protocol 1: Preparation of ES Clone DNA
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Analysis of ES Cell Clones by Southern Blot Hybridization

  Materials
  • Restriction enzyme cocktail (see recipe)
  • 96‐well plate containing ES cell clone genomic DNA (see protocol 3)
  • 10× loading dye (see recipe)
  • 0.8% (w/v) agarose gel in recipeTAE (see recipe)
  • 0.25 M HCl (optional)
  • Denaturing solution (see recipe)
  • Renaturing solution (see recipe)
  • 10× and 5× SSC (see recipe for 20×)
  • 0.4 M NaOH
  • Hybridization solution (see recipe)
  • Radiolabeled and denatured probe
  • 2×, 1×, and 0.1× and SSPE (see recipe for 20×)/0.1% SDS
  • Humidified chamber (e.g., plastic container lined with wet paper towels)
  • Camera
  • Ruler, fluorescent
  • UV transilluminator
  • Whatman paper
  • Hybond N+ nylon membrane (Amersham)
  • Blotting bridge
  • Blotting paper
  • Gel plate or other solid top
  • Weight (∼500 g)
  • 65°C hybridization oven and appropriate hybridization bottle
  • X‐ray film

Alternate Protocol 1: Analysis of ES Cell Clones by PCR

  Materials
  • 96‐well plate containing ES cell clone genomic DNA (see protocol 3)
  • TE buffer, pH 8.0 ( appendix 3C)
  • 10× PCR buffer (supplied with the Taq DNA polymerase)
  • 10 mM dNTPs (2.5 mM each dNTP)
  • Red juice (see recipe)
  • 25 pmol/µl forward and reverse primers
  • 5 U/µl Taq DNA polymerase (Perkin‐Elmer)
  • 0.8% (w/v) agarose or 7.5% (w/v) acrylamide gel (see reciperecipes)
  • 0.5‐ml PCR tubes
  • Thermal cycler

Support Protocol 1: Preparation of ES Clone DNA

  Materials
  • Confluent cultures of ES cells in gelatinized 96‐well feeder‐cell plate (unit 15.1)
  • PBS ( appendix 2A)
  • Lysis buffer (see recipe)
  • 75 mM NaCl in ethanol (see recipe)
  • 70% ethanol
  • Multichannel pipettor
  • Humidified chamber (e.g., a plastic container lined with wet paper towels)
  • 55° to 60°C incubator or hybridization oven
  • Tabletop centrifuge with microplate adapter
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Figures

Videos

Literature Cited

Literature Cited
   Deng, C. and Capecchi, M.R. 1992. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell. Biol. 12:3365‐3371.
   Ramirez‐Solis, R., Rivera‐Perez, J., Wallace, J.D., Wims, M., Zheng, H., and Bradley, A. 1992. Genomic DNA microextraction: A method to screen numerous samples. Anal. Biochem. 201:331‐335.
   Te Reile, H., Maandag, E.R., and Berns, A. 1992. Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc. Natl. Acad. Sci. U.S.A. 89:5128‐5132.
   Udy, G.B. and Evans, M.J. 1994. Microplate DNA preparation, PCR screening and cell freezing for gene targeting in embryonic stem cells. BioTechniques 17:887‐894.
Key References
   Ramirez‐Solis et al., 1992. See above.
   This reference describes a DNA extraction and Southern blotting protocol very similar to that presented in this unit.
   Udy and Evans, 1994. See above.
   Presents a somewhat different 96‐well plate DNA extraction and analysis method.
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