Bio-Rad CHEF Mapper® XA System User Manual

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Conclusion

In these experiments a cDNA probe was used to localize those large restriction fragments

bearing homology to the sequence of interest. Multiple bands were detected with each of the
enzymes used. Numerous situations can give rise to multiple bands on blots with large DNA.
These include the presence of multiple regions homologous to the probe (both genes and
pseudogenes), partial digestion due to restriction enzyme inhibitors in the samples or inter-
ference with cutting by methylation, or allelic differences between chromosomes that make
up the sample. Distinguishing between each of these cases requires knowledge of the factors
which influence pulsed field mobility and application of standard molecular biological meth-
ods to explain the nature of each of the bands.

Fig. 7.1. Separation and identification of fragments smaller than 1 megabase. A. Gel stained with
ethidium bromide. Human DNA in agarose blocks (5 µg/lane) was digested for 6 hr with 40 U of
enzyme in a reaction volume of 150 µl. Following digestion, samples were run in a 1% agarose gel, 6
V/cm for 36 hr in 0.5x TBE at 14 °C. Switch time was a ramp from 55 sec to 75 sec. Samples were:
Lane 1, Yeast size standard (YNN295). Lane 2, Mlu I digest. Lane 3, Nru I digest. Sizes (in kb) are indi-
cated to the left of the yeast standards. B. Autoradiogram showing the position of bands identified by
the probe. After staining, the gel was exposed for 1 minute on a short wave UV light box (254 nm, 2
mW/cm). DNA was denatured in NaOH/NaCI, neutralized with Tris/NaCI and transferred overnight with
10x SSC to Zeta-Probe

membrane according to the manufacturer’s protocol. The hybridization probe

was a cDNA clone for a human G protein b subunit labeled by random priming.

1 2 3

830-

690-

450-

215-