Our current strategy with siRNA is to synthesis relatively small amounts enzymatically and use these to test for efficiency by western blotting. This allows us to test several candidate siRNAs quite cheaply. The yields are good enough for reasonable amounts of experimental work, but for larger use we get successful siRNAs synthesised chemically by Dharmacon. Design siRNA synthesis 5μl oligo 5μl T7 promoter oligo (5'-GGTAATACGACTCACTATAG-3') Snap cool on ice. ii. Small scale transcription reaction 5μl 10x T7 Buffer* 2μl 25mM NTPs (1mM final) 1μl annealed template mix (100nM final) 2μl T7 RNA polymerase (50U/μl) Incubate at 37℃ for 2h. Add 1ul (1U/ul) DNase I (RNase Free) Incubate at 37℃ for 15min |
iii. Generate siRNA duplexes Heat at 95℃ for 5min. Incubate at 37℃ for 1h. Precipitate by adding 6.7ul 3M sodium acetate pH 5.2 + 250ul ethanol. Wash once with 70% ethanol Air dry Dissolve in 50ul water. Day 2 Transfect in late afternoon/evening. Mix siRNA with filter-sterilised 0.25 M CaCl2 then with filter-sterilised BBS (50 mm BES, pH 6.95, 280 mm NaCl, 1.5 mm Na2HPO4). Leave 15-20 min then add dropwise to plates whilst swirling. Remove plate to a 3% CO2 incubator . Day 4 Harvest. General notes |
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