RNAi Plates

Protocol for bacterial mediated RNAi

1. Transform 0.5 ul of RNAi plasmid vector (i.e. L4440, LT61 etc.) into HT115 competent cells.

2. Plate on Amp (50ug/ml) Tet (15 ug/ml) LB plates and grow O/N 37oC.

3. Pick colonies and  inoculate 10ml 2XTY Amp (50ug/ml) Tet (15ug/ml) and grow overnight at 37oC (saturated).

4.  Once saturated store cultures at 4oC. You may also want to freeze down a sample in 15% glycerol at -80oC. RNAi bacterial cultures should only be kept for no more than 1 month at 4oC. New plasmid transformations or inoculations from frozen stock should be made every month or when needed.

5. Prepare RNAi plates by spreading 65ul (flame hockey stick) to each MYOB worm plate:

                        5ul Amp (100mg/ml)

                        5ul IPTG (0.8M)

                        55ul H2O

If we assume there is about 10ml of MYOB on each plate the final concentrations are roughly  50ug/ml Amp, and 0.4mMIPTG (note we leave out Tet on the plates see below).  Make a master mix and scale up for how many plates required. Note the IPTG induction is done on the plate.

 

6. Seed 100ul (flame hockey stick) of saturated bacteria culture on each plate. Don't forget to include an empty vector (L4440) control with every RNAi  experiment. Also a positive control i.e. fem-1 (LT63 or D9) unc-22 (LT61 or D7)  or GFP (L4417 or D11) vectors from the Fire Lab kit. Let seeded plates grow overnight at room temperature and then store at 15oC.

 

7. Pick 5-6 L 4 animals to each plate. Observe progeny for RNAi phenotypes.  It may be necessary to transfer some of the progeny to a new RNAi plate.

 

Other References:

AHRINGER LAB
Clone information and RNAi Feeding Protocol (Version 11.04.01)

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).

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 theCaenorhabditis 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 µg/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 µg/ml final concentration
IPTG to 1 mM final concentration

Plates are poured fresh 1-3 days before use.

Feeding Protocol (from Kamath et al. (2000) Genome Biology, 2, 1-10):

1. Pick and grow bacteria 6 hours - overnight (but no longer than 18 hours) in LB + 50 µg/ml ampicillin, seed onto NGM agar plates including additives (above). (Do not add IPTG or tetracycline to the liquid culture, as this will reduce the RNAi effect.). Bacterial cultures grown shorter times (6 hours) sometimes give better results. The lawn quality is improved if the culture is dried quickly by leaving the lids off for about 20 minutes after seeding, but we don't know if this affects the RNAi effect. We also have anecdotal evidence that plates poured at least 1 week before use work better than freshly poured plates.

2. Let dry and induce overnight at room temperature.

3. The following day, transfer an L4-stage hermaphrodite 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°C (or 36-40 hours at 22°C) for RNAi to take effect, then replica plate adult 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°C vs 22°C; thus, it may be worthwhile to test a given gene using both conditions.