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Male chimeras are test bred to C57BL/6 females and Agouti offspring are genotyped by dot-blot hybridization of tail DNA. We use a rapid one-tube method to prepare genomic DNA. To obtain uniform hybridization signals, it is essential to saturate the membrane with DNA. This protocol was worked out empirically by using a 96-well dot-blot manifold (BioRad) and Hybond N+ hybridization membrane (Amersham). With this method, we can reliably distinguish animals that are homozygous or heterozygous for gene-trap vector insertions based on signal intensity. Either radioactive or nonradioactive probes may be used.
Detailed protocol:
- Prepare fresh tail buffer by dissolving proteinase K powder (Sigma) in 10 mM Tris-HCl (pH 8), 100 mM NaCl, 50 mM EDTA (pH 8), and 0.5% SDS to a final concentration of 1 mg/ml.
- Prepare fresh tail buffer by dissolving proteinase K powder (Sigma) in 10 mM Tris-HCl (pH 8), 100 mM NaCl, 50 mM EDTA (pH 8), and 0.5% SDS to a final concentration of 1 mg/ml.
- Add 0.1 ml of 5 M NaCl to digested tails while still warm and vortex at high speed for 10 seconds. Add 0.5 ml of chloroform and vortex as before. Spin in microfuge at full speed for 15 minutes.
- Transfer 50 µl of the aqueous (top) phase to a 96-well plate. Denature DNA by adding 150 µl of 0.53 M NaOH and incubating for 30 minutes at 37°C.
- Prepare Hybond N+ hybridization membrane by prewetting briefly in water and soaking in 0.4 M NaOH for 10 minutes. Wet one piece of Whatman paper in 0.4 M NaOH and place on the manifold. Lay membrane on top, assemble the apparatus, and briefly apply vacuum to empty wells of excess liquid.
- Apply samples and wait for 30 minutes before applying vacuum.
- Gently vacuum samples through manifold. Disassemble apparatus and wash membrane in 150 mM sodium phosphate buffer containing 0.1% SDS at 65°C for 1 hour.
- Hybridize membrane overnight in Church and Gilbert prehybridization/hybridization buffer (0.5 M sodium phosphate buffer, 7% SDS, and 2.5 mg/ml fraction V bovine serum albumin (Sigma) with a random-prime-labeled ß-geo probe.
- Wash filter three times with 30 mM sodium phosphate buffer containing 0.1% SDS at 60°C and expose filter to autoradiographic film.
§ Excerpted from "Gene Trapping Methods for the Identification and Functional Analysis of Cell Surface Proteins in Mice" by William C. Skarnes in: Methods in Enzymology, volume 328, edited by John N. Abelson, Jeremy Thorner, and Scott D. Emr. Copyright © 2000 by Academic Press. This material may not be reproduced, stored in a retrieval system, or transmitted in any form or by any means without the prior written permission of the publisher.