( pend on thetype and GC content of th...)The DIG System Nonradioactive and,,,Highly Sensitive Detection of Nucleic Acids

pend on the type and GC content of the hybrids. RNARNA and RNADNA hybrids will require higher hybridization temperatures than DNADNA hybrids. In general, the relative strength of different hybrids is RNARNA hybrids > RNADNA hybrids > DNADNA hybrids. As a rule of thumb, for mammalian targets containing 40% GC, the optimal hybridization temperatures in the presence of DIG Easy Hyb or 50% formamide are:

• DNA-DNA: 3742C

• DNA-RNA: 50C

• RNA-RNA: 68C
  

We strongly recommend the use of the DIG EASY HYB buffer, which was specially developed for the DIG System and is nontoxic and guaranteed to be free of contaminants, DNase and RNase.

Amount of target nucleic acid to load on a gel

As a result of the high degree of sensitivity of the DIG System, the amount of target loaded is less than that of comparable systems (Table 2). Note that nylon membranes, positively charged, give the strongest signals and the lowest background when detecting DIG-labeled hybrids.

Detection methods

Probe-target hybrids are usually detected with an alkaline- phosphatase-conjugated antibody either by a color reaction or by a chemiluminescence reaction (Table 3). Of the many alkaline phosphatase substrates that can be used, we recommend CDP-Star or CSPD for chemiluminescence reactions and NBT/BCIP for color reactions.

Detection substrates

• CDP-Star ready to use
  

Detection of alkaline phosphatase activity with CDP-Star results in light emission that can be recorded by e
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