Microplate DNA preparation, PCR screening and cell freezing for gene targeting in embryonic stem cells.
We have developed a new method for genomic DNA microextraction using acetone/N,N-dimethylformamide. Combining this with 96-well plate PCR allows rapid analysis of large numbers of genomic DNA samples. One application is the analysis of embryonic stem cells for rare gene targeting events. The combination of 96-well plate tissue culture procedures, DNA microextraction and PCR reduces the delays and risks associated with Southern analysis and avoids the need to freeze large numbers of cell clones. In contrast to previously described rapid genomic extraction procedures, the DNA produced using acetone/N,N-dimethylformamide microextraction is sufficiently pure to support reliable amplification of at least 2.2 kb. The efficacy of the combined microextraction and amplification procedure has been demonstrated by the identification of gene targeting events at the mOR gene locus. In addition, we have also detailed an improved 96-well plate freezing protocol for embryonic stem cells that allows long-term storage at -70 degrees C. The simple nature of the combined procedures described lend themselves to future automation.
Ruakura Agricultural Centre, Hamilton, New Zealand Wellcome/CRC Institute Cambridge, UK.
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Evans
D000096:Acetone; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification*; D004121:Dimethyl Sulfoxide; D004126:Dimethylformamide; D004622:Embryo, Mammalian; D005312:Fetal Blood; D005615:Freezing; D018390:Gene Targeting / Q000379:methods*; D016258:Genes, mos; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D013234:Stem Cells / Q000737:chemistry*
D000096:Acetone; D004126:Dimethylformamide; D004247:DNA; D004121:Dimethyl Sulfoxide