RFLP Standardization Report for DR Beta/MsPI
Faint bands were listed as standard fragments when they were identified by at least two of the four laboratories.
Faint bands seen by only one laboratory, sometimes in only one lane, were not included. This was seen most often with bands of high kb and may have been due to incomplete digestion.
Discrepancies between blots were resolved in favor of the majority, or in favor of the positive identification when two labs recorded a band and two did not.
Faint bands in particular were sometimes resolved as a doublet in one lab’s blots, but appeared as a singlet in others. Such bands were considered as two standard fragments when they could be consistently resolved on the blots from at least two labs; otherwise they were considered as one band.
Because of small differences in measurement from blot to blot and from lab to lab, the standardized molecular sizes of cross-hybridizing bands were not always exactly the same. In addition, the pattern of positives and negatives of the same band with different cross-hybridizing probes was not always identical. Therefore, cross-hybridizing bands were identified by overlaying the pairs of autoradiographs of blots hybridized with the relevant probes.
KeywordsFaint Band Polymorphic Fragment Marker Band Restriction Element Incomplete Digestion
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