| Stripping and Reprobing Standard methods for removing probes from blots to allow subsequent hybridization with a different probe often include harsh treatments with boiling 0.1% SDS or even autoclaving. These procedures not only damage the membrane, but can also damage the target RNA bound to the membrane, potentially decreasing the signal obtained from the next hybridization. However, probes prepared with one of the Strip-EZ™ Probe Synthesis and Removal Kits can be removed with mild washing conditions, allowing a blot to be stripped and re-hybridized up to 10 times without a significant loss of signal or damage to the membrane. The Strip-EZ Kits include the reagents necessary to synthesize a probe containing a specific modified nucleotide. The modification does not affect the hybridization characteristics of the probe, but enables selective degradation of the probe molecule using a specially designed buffer. In a typical experiment, a StripAble™ probe is synthesized, incubated with a blot, washed, and the probe signal is detected via autoradiography or nonisotopic secondary detection. The blot is then incubated in Probe Degradation Buffer, which cleaves the probe at the site of the modified nucleotides, reducing the RNA or DNA probe to short oligonucleotides. The degraded probe is easily dissociated from its target during the mild degradation wash (10 min., 68°C), allowing the blot to be probed again without residual signal or loss of bound target. Strip-EZ™ StripAble™ Probe Synthesis and Removal Kits are available for the synthesis of DNA probes by random-priming (Strip-EZ DNA Kit) or asymmetric PCR (Strip-EZ PCR Kit) and the synthesis of RNA probes by in vitro transcription (Strip-EZ RNA Kit). |