Microsoft word - rce1182.c-elegans.rnai feeding.clone.doc
C. elegans RNAi Feeding Clones Product description
The C. elegans RNAi Library includes clones derived from the C. elegans ORFeome
Library v1.1 (http://www.openbiosystems.com/c_elegans_orf_clones_release_1_1.php). The Open
Reading Frame (ORF) clones contain the coding sequences located exactly between the
initiation and termination codons, excluding the 5’ and 3’ mRNA untranslated regions (UTRs)1.
Each clone targets a single gene. Cloned ORFs provide an alternative to the genomic DNA
fragments previously described and as ORFs are free of introns, they provide more template
for in vivo siRNA production. High-throughput recombinational cloning protocols were then
used to transfer the C. elegans ORFeome into the pL4440-Dest RNAi feeding vector.
The C. elegans ORF-RNAi Feeding clones are provided as stock cultures of E. coli in
LB broth with an inert growth indicator, 8% glycerol, ampicillin (red cap) at a concentration of
100µg/ml, and tetracycline at a concentration of 12.5 µg/ml.
Clone storage C. elegans ORF-RNAi Feeding clones are shipped at ambient temperature.
The clones may be stored at 4oC for up to one week. Long-term storage should be at -80oC.
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Selectable Marker
The HT115 (DE3) host carries a tetracycline marker conferred by a transposon. The
clones can be grown in ampicillin only since the tetracycline marker is very stable.
The following pairs of universal primers can be used for PCR amplification and
pL4440-dest-RNAi-FOR (5’ GTTTTCCCAGTCACGACGTT 3’)
pL4440-dest-RNAi-REV (5’TGGATAACCGTATTACCGCC 3’)
Verification
The C. elegans ORFeome clones that have been transferred into the RNAi feeding
vector have been end sequence verified. A sampling of 100 RNAi clones has been randomly
picked and sequenced. Ninety-six percent of the RNAi clones contained the expected
sequence. It is strongly suggested that you sequence your clones prior to an experiment if
you use less than 100 RNAi clones. If you perform a genome scale analysis, it is suggested
you sequence verify your “hits” following the RNAi screening.
Making a stock culture
Once the clone has been streak isolated and the identity of the strain has been
confirmed, we recommend making a stock of the pure culture.
1. Grow the pure culture in LB broth plus ampicillin (100 µg/ml) and tetracycline (12.5 µg/ml).
2. Transfer 920µl of culture into a polypropylene tube and add 80µl sterile glycerol to make
3. Vortex the culture to evenly mix the glycerol throughout the culture. The culture can be
Background
The C. elegans ORFeome v1.1 library contains 11,942 ORF clones, comprising
10,623 ORFs cloned “in frame” plus 1,319 ORFs cloned out of frame. ORFs were cloned out
of frame because of mis-predictions of their Start or Stop codons. Only the in-frame ORFs
can be used for protein expression but both sets of clones can be used for RNAi. In all, over
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11,800 RNAi clones were generated. These clones were archived as glycerol stocks of
transformed E. coli strain HT115(DE3) for RNAi feeding protocols and as templates for in vitro
dsRNA synthesis for soaking or injection protocols.
In the C. elegans ORFeome v1.1, each clone represents a mini-pool of PCR
amplified inserts cloned into the pDONR223 vector, not a single unique insert. Each pool is
expected to contain the source ORF, but it is formally possible that various by-products might
have contaminated the pool during the various cloning steps. For example, although PCR
conditions were optimized (high proof reading DNA polymerase and limited number of cycles)
mutations will still occur at low frequency during the PCR amplification. Out of ~70,000 insert
nucleotides sequenced, the mis-incorporation rate was 1 mutation every 35,000 nucleotides.
Following transfer to pL4440, a sampling of 100 RNAi clones have been randomly picked and
sequenced resulting in 96% of the RNAi clones revealing the expected sequence.
Finding further information on clones
The Open Biosystems Clone Query provides a rapid means of locating relevant clone
information. In step 2 of the query, choose the appropriate search criteria from the drop-down
Example of search criteria: Clone ID number: C25E10.11
Figure 2: Open Biosystems Clone Query
In the box for step 3, enter your search list. Click “Begin Search”. Example search criteria
and detailed instructions are available, if necessary, by clicking the question mark icon in the
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upper right corner of the query (Figure 3).
Figure 3: Query Assistance
Clicking the “View” link on the query result page will display the clone information page
containing vector and host information (Figure 4).
Figure 4: Open Biosystems Clone Query Results Useful websites C. elegans genome and RNAi data. http://www.wormbase.org
C. elegans ORFeome data. http://worfdb.dfci.harvard.edu
• Source for C. elegans RNAi data. http://nematoda.bio.nyu.edu
Reference
1 Walhout, A.J., G.F. Temple, M.A. Brasch, J.L. Hartley, M.A. Lorson, S. van den Heuvel,
and M. Vidal. 2000b. GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol328: 575-592
Phone: 1-888-412-2225 FAX: 1-256-704-4849 support@openbiosystems.com
Additional references
Rual, J.F., Ceron, J., Koreth J. , Hao, T., Nicot, A.S., Hirozane-Kishikawa, T., Vandenhaute,
J., Orkin, S.H., Hill, D.E., van den Heuvel, S., and Vidal, M. Towards Improving
Caenorhabditis elegans Phenome Mapping With an ORFeome-Based RNAi Library.
Genome Research 2004 Oct;14(10B): 2162-2168.
Timmons, L. and Fire, A. 1998. Specific interference by ingested dsRNA. Nature 395: 854.
Kamath, R.S., Fraser, A.G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot,
N., Moreno, S., Sohrmann, M., et al. 2003. Systematic functional analysis of the
Caenorhabditis elegans genome using RNAi. Nature 421: 231-237.
Reboul, J., Vaglio, P., Rual, J.F., Lamesch, P., Martinez, M., Armstrong, C.M., Li, S., Jacotot,
L., Bertin, N., Janky, R., et al. 2003. C. elegans ORFeome version 1.1: experimental
verification of the genome annotation and resource for proteome-scale protein
expression. Nat. Genet. 34: 35-41.
Phone: 1-888-412-2225 FAX: 1-256-704-4849 support@openbiosystems.com
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