Bacteria-Mediated (Feeding) RNAi Protocol

Protocol by Min-Ho Lee

Modified by Sudhir Nayak

This protocol is based on: Timmons L, Court DL, Fire. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene. 2001 Jan 24;263(1-2):103-12.

Materials

Double T7 RNA Pol Vector; pPD129.36

E. coli strain expressing T7 RNA polymearase; HT115 (DE3)

Methods

1. Clone a fragment of cDNA into pPD129.36 (use regular competent cells here). Genomic DNA also works but not as well as cDNA.

2. Transform pPD129.36 with your favorite gene cloned into HT115 (DE3).

3. Inoculate 2-4 colonies into 2 ml LB plus 100 ug/ml Amp and 10 ug/ml Tet and grow overnight at 37C rotator.

4. Dilute culture 1:100 into 2x LB plus 100 ug/ml Amp and 10 ug/ml Tet and grow for 6hrs at 37C. We usually grow 5 ml in 15 ml snap cap Falcon tubes, which is good enough to plate about 20 plates at approximately 200ul/plate. You can also make glycerol stock here.

5. Seed bacteria onto plates containing lactose(0.2%) and AMP(100ug/ml) or IPTG(1mM) and AMP(100ug/ml).

6. Incubate at RT for 3 days to grow bacteria and induce dsRNA.

7. Transfer 3-5 worms (L4/young adult) per plate and incubate either 20C or 25C. Transfer the worms to new plates next day. If the RNAi has a strong Po sterile or embryonic lethal phenotype more worms can be seeded.

* Feeding RNAi works at 15C, 20C as well as at 25C. But for some genes, it works better at 25C. I found that the first plates usually have the mixture of wild-type, weak loss of function and strong loss of function phenotypes. The second plates, however, have mostly strong loss of function phenotype, especially at 25C)