Depending on the nature of the experiments, it may be desirable to know the sex of either the embryos used to culture PGCs or of any EG cell lines generated. This can be done easily by using PCR to amplify sequences found only on the Y chromosome (16,22) (see Note 8).
1. To generate DNA samples from embryos or cell lines, tissue is incubated in lysis buffer with 0.5 mg/mL proteinase K overnight at 56°C. On the next day, samples are extracted once with phenol, once with phenol:chloroform:isoamyl alcohol (25:24:1), and once with chloroform:isoamyl alcohol (24:1), and then ethanol-precipitated. References for these molecular biology techniques can be found elsewhere in this volume or in ref. 23.
2. PCR reactions are performed in a temperature cycler with 400 ng of genomic DNA as a template. Reactions are set up as follows:
5 |L dNTPs (10 mM each nucleotide).
5 |L 10X PCR buffer.
1 |L each primer.
3. Reactions are cycled at 95°C for 45 s, 62°C for 25 s, and 72°C for 1 min for a total of 30 cycles.
4. Run 1/10 or more of the completed reaction on a 1% agarose gel in order to detect the presence of a 600-bp reaction product indicating the presence of a Y chromosome.
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