There are several problems that may occur that will result in the failure of a signal to appear on the final blot. To help eliminate possibilities, consult the following checklist:
Was enough DNA loaded onto the gel (10 p,g)7 Was the DNA digested thoroughly prior to electrophoresis? Was the electrophoresed DNA denatured and neutralized before transfer?
Was transfer complete? (Stain the gel after transfer to assess residual DNA.)
Was the DNA immobilized properly onto the membrane support?
Was the DNA intact? (Has the membrane been stripped and probed multiple times?)
Was the probe prepared properly?
Was the hybridization time sufficient (at least overnight)?
Was the blot exposed to film for a sufficient amount of time?
Was the probe labeled sufficiently?
If the answer to all of the above questions is "yes," then the failure of a positive signal to appear could be related to the strength with which the probe hybridized to the target sequences on the membrane. If the probe is from a species other than the test DNA, weak hybridization might be the result of a lack of homology between the probe and the target gene sequence. Decreasing the stringency of the posthy-bridization washes and/or increasing the length of exposure time while generating the autoradiograph can often strengthen a very weak signal. Alternatively, it might be necessary to try a different probe or a different probe-labeling technique.
High background on the final blot is usually indicative of insufficient blocking during the prehybridization and hybridization steps or of poor washing of the blot after hybridization. To eliminate the background, simply increase the amount of denatured salmon sperm DNA added to the pre-hybridization and hybridization solutions and/or increase the stringency of the posthybridization washes by decreasing salt concentration or increasing temperature. When RNA probes (riboprobes) are utilized, the temperature of hybridization washes might need to be as high as 65°C to eliminate background hybridization.
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