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)