Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4415

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Genotyping Embryonic Stem (ES) Cell Colonies Prior to Picking

Philippe Soriano

This protocol was adapted from "Introduction of Foreign DNA into Embryonic Stem Cells," Chapter 10, in Manipulating the Mouse Embryo, 3rd edition, by Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

The first 15% of the full text of this article appears below.


INTRODUCTION

This protocol describes a rapid PCR-based method for identifying targeted ES cell colonies prior to picking. It is based on DNA analysis of a small part of colonies pooled directly from selection plates. Only positive colonies are expanded.


MATERIALS

Reagents

AmpliTaq polymerase (Perkin Elmer)

Bovine serum albumin (BSA) (nuclease-free; Boehringer Mannheim; stock 1.6 mg/ml)

caution Dimethyl sulfoxide (DMSO) (Aldrich)

recipe DNA in ES PCR Lysis buffer (include positive and negative controls)

dNTPs (Pharmacia; stock of the mix 10 mM)

ES colonies in G418 selection (see Electroporating DNA into . . . [Full Text of this Article]

Equipment


METHOD


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Related Protocol

Electroporating DNA into Embryonic Stem (ES) Cells and Selection Methods
Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer
CSH Protocols 2006: 4409. [Extract] [Full Text]