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8. PCR. With SAGE, achieving the initial 102-bp ditag PCR product takes time. Thereafter,
however, equally strenuous efforts are necessary to avoid PCR cross-contamination, hence
the inclusion of negative controls. The Johns Hopkins protocol now recommends three
separate PCR areas, for pre-PCR assembly of reagents and negative controls; the addition
of ligated ditag template; and the manipulation of post-PCR products. Ultraviolet PCR
preparation hoods may also be useful.
9. Polyacrylamide gel electrophoresis (PAGE). The SAGE PCR products and ditags are isolated
by 12% PAGE and the concatemers are separated by 8% PAGE. 12% PAGE uses 14 mL
of 40% polyacrylamide (191 acrylamidebis) and 31.3 mL of dH 2 0. 8% PAGE requires
9.3 mL of 40% Polyacrylamide (37.51 acrylamidebis) and 36 mL of dH 2 0. To either,
add 930 µL of 50× Tris acetate buffer, 470 µL of 10% ammonium persulfate, and 30 µL of
TEMED. Mix and pour in a vertical gel apparatus. Allow to polymerize for 30 min. Run
gel as described in the text (the time is approximate). (Note that bromophenol blue is dark
blue and runs ahead of xylene cyanol, which is turquoise.) After electrophoresis, stain gel
with SYBR ® Green I, according to manufacturer’s instructions, or with ethidium bromide.
Visualize bands under ultraviolet light. The Johns Hopkins and I-SAGE protocols and some
of the methodological papers contain useful photographs of sample gels at each stage.
10. DNA quantitation. The Johns Hopkins and I-SAGE protocols recommend assessment
of DNA yield by dot quantitation at various stages during PCR purification and ditag
isolation. In general, however, this is not routinely required.
Acknowledgments
Thanks to the Cancer Research Campaign and the University of Glasgow for research
funding and to Dr. Kenneth W. Kinzler for SAGE protocols, software and advice.
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