Clone information and RNAi Feeding Protocol

AHRINGER LAB
Clone information and RNAi Feeding Protocol

Vector and inserts:

Genomic fragments obtained by PCR were cloned into the Timmons and Fire feeding vector (L4440), which is a modified version of Bluescript with a T7 promoter on each side of the MCS driving transcription of each DNA strand (Nature, 395, 854). Information about the L4440 vector (including sequence information) can be found at http://www.ciwemb.edu. PCR fragments were obtained using Research Genetics GenePairs. The GenePairs primer sequences are available at http://cmgm.stanford.edu/~kimlab/primers.12-22-99.html and are displayed visually in WormBase (http://www.wormbase.org). Clones are available from MRC geneservice (http://www.hgmp.mrc.ac.uk/geneservice/reagents/products/rnai/index.shtml).

Bacteria:

Genomic fragments cloned into L4440 were transformed into HT115 (DE3), an RNase III-deficient E. coli strain with IPTG-inducible T7 polymerase activity (Gene, 263, 103-112). The strain is available from the Caenorhabditis Genetics Center (http://www.cbs.umn.edu/CGC/CGChomepage.htm). The genotype is as follows: F-, mcrA, mcrB, IN(rrnD-rrnE)1, lambda -, rnc14::Tn10(DE3 lysogen: lavUV5 promoter –T7 polymerase) (IPTG-inducible T7 polymerase) (RNase III minus). This strain grows on LB or 2xYT plates (and is resistant to tetracycline, see below), and competent cells can be made using standard techniques.

Note on tetracycline:
The Tn10 transposon interrupting the rnc14 gene carries a tetracycline resistance gene. Therefore, bacteria should be subjected to tetracycline selection (12.5 mg/ml) to maintain the RNase deficiency. However, the transposon is quite stable, as we have not lost it in the absence of selection. Using our protocol (see below and Kamath et al. Genome Biology, 2, 1-10) inclusion of tetracycline during feeding significantly decreased the RNAi effect for several genes tested, so we recommend that it not be used in culture or in NGM plates during feeding using the method below. However, using the method of Timmons, et al (Gene, 263, 103-112), an improvement in feeding results by including tetracycline was reported.

NGM Media:

For feeding plates, use standard NGM agar plus the following ingredients:

Carbenicillin to 25 mg/ml final concentration
IPTG to 1 mM final concentration

Plates are poured 4-7 days before seeding.

Feeding Protocol (from Kamath et al (2001) Genome Biology, 2, 1-10):

1.Pick and grow bacteria 6-12 hours in LB 50 mg/ml ampicillin (until a fairly dense culture is obtained), then seed dropwise onto NGM agar plates including additives above. Use quite a lot of bacteria to inoculate the culture. (Do not add IPTG or tetracycline to the liquid culture, as this will reduce the RNAi effect.).

2.Let dry and induce overnight at room temperature.

3.The following day, transfer L3-L4-stage hermaphrodites onto first plate, minimizing the amount of OP50 bacteria transferred (we usually wash worms in M9 buffer and then aliquot them directly onto plates). Leave 72 hours at 15℃ (or 36-40 hours at 22℃) for RNAi to take effect, then replica plate single adults onto another plate seeded with the same bacteria. After 24 hours, remove the adult from the replica and score the progeny for phenotypes. Alternatively, aliquot embryos or larvae onto the feeding plates and score them later.

4.Note on temperature:
We have observed that some genes give different phenotypes at 15℃ vs 22℃; thus, it may be worthwhile to test a given gene using both conditions.

If you have any questions, please contact Julie Ahringer (jaa@mole.bio.cam.ac.uk).