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)
