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C. elegans RNAi Collection (Ahringer)

C. elegans RNAi Collection (Ahringer)

The Caenorhabditis elegans RNAi feeding library was constructed by Julie Ahringer's group at The Wellcome CRC Institute, University of Cambridge, Cambridge, UK and is distributed by Source BioScience. It is designed for genome wide study of gene function in C. elegans through loss of function studies. It can be used both for rapid large scale RNAi experiments and for the study of individual genes.

When produced initially the library targeted 72% of C.elegans genes (according to current gene annotation). Julie Ahringer and her team in conjuction with Source BioScience have since generated a collection of new supplemental bacterial strains to enhance this library, and the total library now targets around 87% of currently annotated C.elegans genes. These supplemental strains are now available to order from Source BioScience.

The library is available as individual chromosome sets (I, II, III, IV, V and X) which include the new supplemental strains.

The complete supplemental RNAi set of plates and individual chromosome sets (I, II, III, IV, V and X) are also available to order for those who have already purchased the library in the past.

The complete C.elegans RNAi database including the new supplementary bacterial strains can be downloaded here

The following subsets are also available:

Chromatin

Transcription Factors

Phosphatase

 

 

As part of our sequence verification service, individual clones are end sequenced before they are sent to you. If the requested clone does not match the expected sequence, Source BioScience will not apply any charge and will contact the purchaser to confirm if they wish to cancel the order.This risk free option ensures that you have the right clone, removing the chance of receiving one of the wrong clones that are inherent in some libraries and can also save you time from having to sequence the clone yourself.

PLEASE NOTE –that Source BioScience excludes any warranty pertaining to the accuracy of the sequence for clone orders where the client chooses to reject the offered sequence verification option, as the clones are supplied as-is, direct from the original library.

Set details

The library is provided as frozen glycerol stocks of bacterial strains arrayed in 384 well plates.

The complete C.elegans RNAi database including the new supplementary bacterial strains can be downloaded here

The complete set, including the supplemental plates contains the following plates:

Library sets Number of plates Approximate clone number
C.elegans Chr.I RNAi 10 2,875
C.elegans Chr.II RNAi 11 3,594
C.elegans Chr.III RNAi 9 2,630
C.elegans Chr.IV RNAi 10 3,221
C.elegans Chr.V RNAi 16 4,716
C.elegans Chr.X RNAi 8 2,726
C.elegans Chr RNAi - complete set 64 19,762



NEW supplementary sets Number of plates Approximate clone number
C.elegans supplementary Chr. RNAi - Chr.I 2 430
C.elegans supplementary Chr. RNAi - Chr.II 2 620
C.elegans supplementary Chr. RNAi - Chr.III 2 498
C.elegans supplementary Chr. RNAi - Chr.IV 2 528
C.elegans supplementary Chr. RNAi - Chr.V 3 1,060
C.elegans supplementary Chr. RNAi - Chr.X 1 371
C.elegans supplementary Chr. RNAi complete set 12 3,507

 

Protocols / Clone Handling

Please note that the clone(s) are tested as negative in our phage contamination assay prior to shipping. However, these clones should still be handled with care as no phage assay can be guaranteed to be 100% accurate.

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 the Caenorhabditis Genetics Center (http://www.cbs.umn.edu/CGC/). 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.

If you have any questions, please contact Julie Ahringer (ja219@cam.ac.uk)

References

Subsequent publication

Please acknowledge the originator of the library, Dr Julie Ahringer, and Source BioScience for providing the library in any publication arising out of using this resource.

Devgen Notice

  1. The RNAi feeding library may be provided to not-for-profit and commercial entities, but for research purposes only.
    For any intended commercial use of the RNAi feeding technology, your attention is drawn to the following patent applications and patents in the name of Devgen N.V., Belgium: EP-A-1 093 526, EP-A- 1 197 567, GB 2349885-B, GB 2370275-A and GB-2362885-B, and further patent applications and patents that may correspond thereto.
    Source BioScience LifeSciences does not have the right to grant any license (either explicit or implied) under any patent applications and/or patents held by Devgen N.V.. Should you wish to obtain such a license, you are advised to contact Devgen N.V., tel. Belgium-9-3 24 24 24; fax Belgium-9-3 24 24 25 or e-mail: info@devgen.com .
  2. The RNAi feeding library is provided for research purposes only, and may not be used for any commercial purposes, including but not limited to: (1) screening by any entity other than a not-for-profit organization of the RNAi feeding library or any substantial part thereof of on average more than 30 genes per screening day; and/or (2) offering and/or providing to third parties, in return for any financial and/or other consideration, of any services relating to and/or involving the RNAi feeding library.For any intended commercial use of the RNAi feeding technology, your attention is drawn to the following patent applications and patents in the name of Devgen N.V., Belgium: EP-A-1 093 526, EP-A- 1 197 567, GB 2349885-B, GB 2370275-A and GB-2362885-B, and further patent applications and patents that may correspond thereto.Source BioScience LifeSciences does not have the right to grant any license (either explicit or implied) under any patent applications and/or patents held by Devgen N.V.. Should you wish to obtain such a license, you are advised to contact Devgen N.V., tel. Belgium-9-3 24 24 24; fax Belgium-9-3 24 24 25 or e-mail: info@devgen.com

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