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'''Multiplicity controls:''' a good way to enhance confidence in RNAi data is to demonstrate a similar effect with two or more siRNAs targeted to different sites in the message under study. Alternatively, the RNAi approach is usefully supplemented by alternative methods, such as those described above. | '''Multiplicity controls:''' a good way to enhance confidence in RNAi data is to demonstrate a similar effect with two or more siRNAs targeted to different sites in the message under study. Alternatively, the RNAi approach is usefully supplemented by alternative methods, such as those described above. | ||
==siRNA related buffer and solutions== | |||
===RNase-free H2O=== | |||
* 0.1% DEPC in distilled H2O. Mix and leave at room temperature for 1 hour. | |||
* Autoclave | |||
* Cool to room temperature prior to use. | |||
0.1% DEPC inactivates RNase contamination from common environmental sources and laboratory procedures. Autoclaving alone will inactivate a substantial amount of RNase as well. | |||
Autoclaving inactivates DEPC by causing hydrolysis of diethylpyrocarbonate to yield CO2 and EtOH. DEPC has a half-life of ~30 minutes in water; 0.1% DEPC solution autoclaved for 15 minutes/liter can be assumed to be DEPC-free. | |||
===TE Buffer=== | |||
10X TE Buffer (siRNA dilution buffer) | |||
* 10 mM Tris, bring to pH 7.5 with HCl | |||
* 1 mM EDTA | |||
* RNase free water (DEPC water) | |||
The EDTA in TE chelates Mg2+ and other divalent metals ions necessary for most causes of DNA and RNA degradation, suppressing these processes. Each EDTA molecule chelates one Mg2+ ion. | |||
DNA is stored at pH 8 to reduce depurination, which is acid catalyzed, while RNA is stored at a slightly lower pH (7.5) because degradation of RNA is base-catalyzed. Most downstream reactions will not be influenced by the slightly different pH storage conditions. | |||
==References== | ==References== |
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