![]() ![]() In case the probe is not completely removed, any residual signal should not yet appear during the shorter exposure times needed for more abundant probes.Īmbion's Strip-EZ™ Technology, however, renders harsh stripping protocols as described above obsolete. As a precaution, we recommend probing first for the mRNA you expect to be least abundant. The harsh stripping conditions recommended above are only possible because of the extremely high retention of the recommended positively charged nylon membrane. Another important drawback is that these stripping conditions will damage the RNA bound to the membrane after 3-4 strippings. The chief drawback to using RNA probes is that they are difficult to strip due to their inherent high thermodynamic stability when hybridized to RNA targets. Traditional procedures involve either boiling or autoclaving (wet cycle) for ten minutes in a solution of 0.1% SDS in DEPC-treated water, then cooling to room temperature. Membranes can be stripped only if they have never been allowed to dry to completion after hybridization. The final, stringent wash is generally performed at the hybridization temperature in 0.1X SSC / 0.1% SDS or its equivalent. This will usually be 10☌ to 20☌ higher than for a randomly primed DNA probe used to detect the same mRNA target. Hybridization is performed at 15° to 25☌ below the calculated Tm. To approximate the Tm for RNA:RNA duplexes, use the following formula: Ambion's ULTRAhyb Hybridization Solution has incorporated all of these recommendations and has been rigorously tested for nuclease activity. ![]() We have found the addition 100 µg/ml total yeast RNA (total yeast RNA performs better than tRNA) helpful in reducing background and cross-hybridization. Nucleic acid blocking agents must be RNase-free as well. Use only deionized, molecular biology grade formamide. Salt conditions (SSC or SSPE) between 500 mM and 1 M and SDS between 0.1% and 10% have been observed to provide good results (4). Avoid the use of products derived from animal sources, in particular BSA and "BLOTTO". When preparing hybridization buffer, use only reagents known to be RNase-free. The hybridization solution should be formamide-based in order to lower the Tm of the RNA:RNA duplex to a reasonable temperature for most RNA probes this will be around 60° to 65☌. As a result, the hybridization buffer and temperature need to be adjusted to ensure optimal hybridization to target and minimal nonspecific hybridization of probe, particularly to the ribosomal sequences. RNA:RNA duplexes have a higher Tm and binding affinity than do RNA:DNA duplexes. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |