Sigma is both a member and distributor of The RNAi Consortium for phase 1 and 2 (TRC1 and TRC2) development of human and mouse lentiviral-based shRNA libraries. The TRC1 shRNA collection, comprised of over 150,000 clones targeting greater than 15,000 human and 15,000 mouse genes, is complete and clones are available as whole genome libraries, subsets categorized by gene family or pathway, or as individual clones. The TRC2 project is in process and will expand upon the base TRC1 library, inquire with your local Research Biotech Sales Representative for more details.
Sigma is the exclusive provider of TRC clones in purified DNA and lentiviral formats for any combination of TRC clones. Bacterial glycerol stock format is also available. Click on the diagram below to find the product that best meets your needs.
General Information About MISSION TRC shRNA Clones
Each MISSION shRNA clone is constructed within the lentivirus plasmid vector pLKO.1-Puro followed by transformation into Escherichia coli. The pLKO.1–Puro vector contains bacterial (ampicillin) and mammalian (puromycin) antibiotic resistance genes for selection of inserts in either bacterial or mammalian cell lines. Each gene is represented by multiple individual shRNA constructs or clones targeting different regions of the gene sequence. Therefore, a range of knockdown efficiency, with at least one construct from each gene set being >70%, can be expected when using these clones. This allows one to examine the effect of loss of gene function over a range of gene knockdown efficiencies. Each shRNA construct has been cloned and sequence verified to ensure a match to the target gene. The clones are available in frozen bacterial glycerol stock format, purified plasmid DNA format, or lentiviral transduction particle format.
As shown in the RNAi workflow and the diagram below, bacterial stocks may be used to create plasmid DNA. The plasmid DNA may either be transfected directly into your cells of interest or used to make lentiviral particles via a viral packaging process. The DNA and viral formats require less handling and reduce the number of steps to start your research. You may acquire MISSION TRC shRNA clones in any format and start the process at the point that is most convenient to you.
Bacterial Glycerol Stock Format (SHCLNG)
Bacterial cultures may be amplified from the glycerol stocks for use in purification of shRNA plasmid DNA. Subsequently, target cell lines may be transfected with the purified plasmid for transient or stable gene silencing (puromycin selection). In addition, self-inactivating replication incompetent viral particles can be produced in packaging cells (HEK293T) by co-transfection with compatible packaging plasmids.
Individual shRNA clones are provided as a frozen bacterial glycerol stock (E. coli) containing Terrific Broth (TB), carbenicillin at 100 µg/ml and 15% glycerol.
Search for Your Favorite Gene
Go to the Getting Started Page
Download the SHCLNG Technical Bulletin (171 Kb PDF)
Order prior to April 25, 2009? Download the SHGLY Technical Bulletin (590 Kb PDF)
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Purified Plasmid DNA Format (SHCLND)
Sigma offers the highest quality purified plasmid DNA for downstream transfection and lentiviral packaging. DNA purified with Sigma’s 96-well DNA purification system, developed specifically for the TRC library, yields the highest quality lentiviral particles.
Purified plasmid may be directly used in transient or stable gene silencing (puromycin selection). In addition, self-inactivating replication incompetent viral particles can be produced in packaging cells (HEK293T) by co-transfection with compatible packaging plasmids.
Individual constructs are provided as 40 µL frozen stocks containing 1 µg of plasmid DNA in Tris-EDTA (TE) buffer at a concentration of 25 ng/µL. See gene family set technical bulletins for specifications, inquire for library specifications.
Log in and try out Sigma's powerful MISSION shRNA ordering interface to select the standard vector backbone or customize your shRNA by choosing an alternate vector backbone. Search for Your Favorite Gene to try it out! Visit the Vector map website for details on these alternate vectors.
Go to the Getting Started Page
Download the SHCLND Technical Bulletin (149 Kb PDF)
Order prior to April 25, 2009? Download the SHDNA Technical Bulletin (567 Kb PDF)
Lentiviral Format (SHCLNV)
Self-inactivating replication incompetent viral particles are produced in packaging cells (HEK293T) by co-transfection with compatible packaging plasmids. In addition, the lentiviral transduction particles are pseudotyped with an envelope G glycoprotein from Vesicular Stomatitis Virus (VSV-G), allowing transduction of a wide variety of mammalian cells. The lentiviral transduction particles are titered via a p24 antigen ELISA assay and pg/ml of p24 are then converted to transducing units per ml using a conversion factor.
Individual constructs are provided as a 200 µL frozen stock containing a minimum of 1.0 x 106 TU/ml (via p24 assay) in Dulbecco’s Modified Eagle’s Medium with 10% heat-inactivated fetal bovine serum and penicillin-streptomycin. Sigma’s average titers for individual clone orders are routinely greater than 1.0 x 107 TU/ml (via p24 assay). See gene family set technical bulletins for specifications, inquire for library specifications.
Choosing Your Titer and Volume
As a logged in customer, you have the ability to customize not only the vector backbone (visit the Vector map website for details), but also the titer and volume you desire. Please refer to the table below for standard aliquotting. If you require different aliquotting, please use our custom request form.
| |
106 |
107 |
108 |
109 |
| 0.1 ml |
N/A |
0.1 ml |
0.1 ml |
0.1 ml |
| 0.2 ml |
0.2 ml |
0.2 ml |
4 x 50 µl |
10 x 20 µl |
| 1 ml |
5 x 0.2 ml |
5 x 0.2 ml |
20 x 50 µl |
50 x 20 µl |
| 2 ml |
10 x 0.2 ml |
10 x 0.2 ml |
40 x 50 µl |
100 x 20 µl |
| 5 ml |
25 x 0.2 ml |
25 x 0.2 ml |
100 x 50 µl |
N/A |
| 10 ml |
50 x 0.2 ml |
50 x 0.2 ml |
200 x 50 µl |
N/A |
Search for Your Favorite Gene
Go to the Getting Started Page
Download the SHCLNV Technical Bulletin (181 Kb PDF)
Order prior to April 25, 2009? Download the SHVRS Technical Bulletin (123 Kb PDF)
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Manufacturing Information and Shipping Timelines
Orders of 5 or fewer individual clones are provided in individual vials. Each vial contains a unique two dimensional barcode label that can be read using a corresponding reader. A printed value corresponding to The RNAi Consortium (TRC) clone number is also provided on each tube. Orders of >5 individual clones are provided in a 96-well plate with a one dimensional barcode label on the plate. 96-well plates are provided with a CD containing plate map positions.
Each set of individual clones is produced on demand in our state-of-the-art manufacturing facility. The following times are required to produce each format after the order appears in our LIMS manufacturing database:
SHCLNG – Bacterial Stock
|
48 hours |
| SHCLND – Purified DNA |
2 weeks |
| SHCLNV – Lentiviral Transduction Particles |
4 weeks |
Please Note:
If your order includes customization of volume, titer, or vector, the turn-around-time will be longer than listed above.
Additionally, if your order contains >25 individual clones with vector customization, or >50 individual clones in the standard vector, the turn-around-time will be longer than listed here. We recommend contacting your local sales representative for pricing and timelines for larger orders.
Orders ship Monday through Thursday. Glycerol format orders received after 3 pm on Wednesday may not ship until the following Monday. As indicated, some orders may require additional time to process. Please allow up to 2 weeks additional shipping time for international orders.
For questions about the library, pricing and quotes or other concerns, please e-mail us at: RNAi@sial.com.
MISSION is a registered trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co. Label License.
