This standard is based on DdeI and CfoI restriction digestion of the phage PhiX174 (available in its circular, replicative form from Promega, approximately US$75 for 50µg). The 5' overhangs are polished using the Klenow fragment of DNA Polymerase I, and the fragments labeled using Texas Red-dUTP (Molecular Probes). Because of the distribution of A in the 5' overhangs, most DNA fragments are labeled on only one strand.
1. Digest 50µg of PhiX174 DNA with DdeI and CfoI. These enzymes have quite different reaction conditions, so the digests must be done sequentially, with an ethanol precipitation following the first digestion. Use 100µg/ml Bovine Serum Albumin (BSA) in a total reaction volume of 200µl. Use twice the recommended amount of enzyme (they're relatively cheap) and digest for at least four hours. It is recommend that each digest is checking by running on a 2% agarose (4µl of the 200µl reaction). It is very important to have a complete digests of the DNA.
2. Following the second restriction digest, add
80 µl dATP, dCTP, dGTP, (2.5 mM each)( No dTTP!!!!)
8 µl of 1 mM 12-Texas Red dUTP (Molecular Probes), or any other dUTP or dTTP with a fluorescent label
8 µl Klenow Fragment of DNA Polymerase (40 units)
3. Incubate at room temperature for 20 minutes. This is important, because longer incubations can result in poor labeling due to 3' to 5' exonuclease back filling activity of Klenow. The quantities of reagents in the polishing reaction have not been optimized and are likely to be in excess - it may be worthwhile optimizing the reaction concentrations
4. Inactivate the Klenow at 65°C for 10 min.
5. Phenol extraction, ethanol precipitation and resuspend the bring pellet in 1.5mL of 10/1 TE (10 mM Tris, 1mM EDTA, pH 8.0).
6. Use about 0.3µl per lane on the ABI 377 (even less with the 3700 or 3730). This preparation should be adequate for a minimum of 5000 lanes. You'll need to try it out first, of course. Yields will vary depending on the efficiencies of the extractions. Also, the sensitivity of the fluorimager to the fluorescent dye will depend on the wavelength of the excitation laser and the collection filters. For example, the ABI377 is about ten times more sensitive to FAM than to Texas Red, and the FMBIOII is more sensitive to Texas Red than to FAM.
7. Expected sizes of labeled DNA fragments (bp):
52*, 81, 113 ,117, 158, 186, 192, 217, 277, 312, 328, 340, 486*, 490, 577, 599
Asterisks indicate fragments labeled on both strands.
The standard works well, except for the 186, 312, and 340 fragments which migrate slightly outside the expected range. It is therefore advised to exclude these fragments from the analyses.