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Load the DNA standard size marker on the gel. 6. Run the gel in 1 x TAE buffer at 80 V. Stain the gel with 50 ml of 1 x TAE buffer plus 5 (j,I SYBR Green 1 dye or with ethidium bromide. Visualize the DNA bands with long wavelength, low frequency, UVB light, or scan the agarose gel with the GenomyxSC Imager. If there are other bands in addition to the target band, it will be necessary to purify the appropriately sized band via SSCP (Protocol 7) before it is used in DNA sequencing, ribonuclease protection assays (RPA), or Northern blot screening.
Mineral oil overlays can be used in the event that a thermal cycler with a heated lid is unavailable, but the results may be less consistent. ) Sterile nuclease-free H2O(E476, Amresco: 9920. Ambion; or DEPC-treated H2OK) Method 1. Thaw out the reagents'1 (first strand cDNAs, TMR 5' end-labelled fluoroDD APs corresponding to the HIEROGLYPH APs used in the RT samples produced in Protocol 2, imlabelled HIEROGLYPH ARPs, dNTP mix, 10 x PCR buffer II, MgCl2 stock solution, and AmpliTaq DNA polymerase) on ice, and keep them on ice.
Guimaraes, M. , Bazan, J. , Wiles, M. , Grimaldi, J. , et al. (1995). Development, 121, 3335. 4. , and Spiegelman, B. J. Biol. , 271,10697. 5. Babity, J. , Newton, R. A, Guide, M. , and Robertson, H. A. (1997). In Methods in molecular biology: differential display methods and protocols (ed. P. Liang and A. B. Pardee), p. 285. Humana Press, Totowa, NJ. 6. McCarthy, S. , Samuels, M. L, Pritchard, C. A, Abraham, J. A, and McMahon, M. (1995). , 9,1953. 7. Linskens, M. H. , Andrews, W. , Enlow, B. , Saati, S.