Here is our T7 siRNA protocol. The main idea is to design primers that begin with GG so that transcription by T7 polymerase is efficient. I) FIRST design oligos: Using sense coding sequence of any gene.... (N1, N2, N22, N23, are numbered positions) 1) Find 5'-N1 N2 G/A G/A NNNNNNNNNNNNNNN C C N22 N23-3' 2) Drop N22 and N23 add 5'-TATAGTGAGTCGTATTA-3' to 3' END to get 5'-N1 N2 G/A G/A NNNNNNNNNNNNNNN C C TATAGTGAGTCGTATTA-3' = oligo A 3) From (1) 5'-N1 N2 G/A G/A NNNNNNNNNNNNNNN C C N22 N23-3' drop N1 N2 convert G/A G/A to G G to get 5'-G G NNNNNNNNNNNNNNN C C N22 N23-3' 4) Add 5'-TAATACGACTCACTATA-3' to 5'END to get 5'-TAATACGACTCACTATA G G NNNNNNNNNNNNNNN C C N22 N23-3' 5) Get reverse complement of (4) 5'-N23' N22' G G NNNNNNNNNNNNNNN' C C TATAGTGAGTCGTATTA-3' = oligo B 6) Rank the initial target sequences with GG preferable to G G/A preferable to G/A G with A A not even considered unless absolutely necessary. GG N15 CC > G G/A N15 CC > G/A G N15 CC >>>>>>>>> A A N15 CC 7) Order oligos II) THEN make RNA in vitro 8) Anneal oligo A and oligo B separately to T7 primer (TAATACGACTCACTATAGG) or toprimers that are complementary to oligoA and B. 9) Use 2-3 ug annealed primer in a 20ul Ambion T7 Megashort script reaction Ambion Cat# 1354 (Follow Ambion’s protocol Incubate 2-4 hrs) 10) Combine oligo A and B reactions 11) Anneal T7 transcribed RNAs using your favorite slow annealing protocol e.g. 95℃ 5 min 70℃ 5min 50℃ 5min 37℃ 5min 12 A) Phenol-chloroform extract annealed, transcribed RNAs / Ethanol precipitate / 12 B) Or Purify on Ambion MegaClear columns 13) Resuspend in 50-250ul H2O 14) Quantify yield 15) Transfect use at least 0.5ug per well of a 6 well plate (3.0ug per 10 cm dish) |
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