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Kafatos:dsRNA Production by PCR: Difference between revisions
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Kafatos:dsRNA Production by PCR (view source)
Revision as of 04:51, 10 October 2008
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=== Purification of the RNA === | === Purification of the RNA === | ||
There are two steps for the purification of the dsRNA: the digestion of the DNA template and the clean-up of the sample with the '''RNeasy kit (QIAGEN)'''.<BR> | There are two steps for the purification of the dsRNA: the digestion of the DNA template and the clean-up of the sample with the '''RNeasy kit (QIAGEN)'''.<BR> | ||
(β-Mercaptoethanol (β-ME) must be added freshly to the Buffer RLT before use (under hood!). Add 10 μl β-ME per 1 ml Buffer RLT – you only need 350 μl Buffer RLT per reaction. Also make sure that 4 volumes of ethanol have been added to 1 volume of Buffer RPE.)<BR> | NOTE: The addition of β-Mercaptoethanol is not necessary and should be avoided. (β-Mercaptoethanol (β-ME) must be added freshly to the Buffer RLT before use (under hood!). Add 10 μl β-ME per 1 ml Buffer RLT – you only need 350 μl Buffer RLT per reaction. Also make sure that 4 volumes of ethanol have been added to 1 volume of Buffer RPE.)<BR> | ||
• ''Digestion of the DNA with DNAseI:'' add 1μl DNAse (Ambion Kit) to the 20 μl reaction and incubate at 37˚C for 15 min.<BR> | • ''Digestion of the DNA with DNAseI:'' add 1μl DNAse (Ambion Kit) to the 20 μl reaction and incubate at 37˚C for 15 min.<BR> | ||
