We use the Promega Ribomax Large Scale RNA Production System T7 (Cat. No.: P1300) to produce dsRNAs and the RNAi Exp.html" target=_blank< Jack Dixon protocol ( RNAi _Dixon.html" target=_blank
I. Preparation of Template DNA
1.Design two oligos for your gene of interest.
Each should incorporate a 5' T7 RNA polymerase binding site, resulting in a PCR product of approximately 700 bp.
2.T7 RNA polymerase binding site: TTA ATA CGA CTC ACT ATA GGG AGG
3.Purify the PCR DNA template
It should be free of RNases and inhibitors such as high salt, detergents or EDTA.
4.Quantify the PCR Product on an agarose gel.
The dsRNA reaction detailed below requires 5-10 µg total DNA in 40 µl.
II. Preparation of the dsRNA
We use the Promega Ribomax Large Scale RNA Production System T7. We modify the supplied protocol as follows:
1.Prepare the templates as described above.
2.Add the following reagents from the kit in a 1.5 ml Eppendorf tube at RT. For a 100µl reaction:
20 µl Buffer 5x
30 µl rNTPs (25 µM)
DNA 5 µg
10 µl T7 enzyme mix
nuclease free water to a final volume of 100 µl
3.Mix tube gently, spin contents down and incubate at 37
