Silencer庐 Human Kinase siRNA Library Product Information Sheet
庐
Silencer Human Kinase siRNA Library
Product:
AM80010V3
Catalog #:
AMO07XDS
Lot #:
0.25 nmol each siRNA
Amount:
2157 unique siRNAs targeting each of 719 kinase genes
Content:
Total of 27 96-well plates (plates are Axygen #PCR96FS; www.axygen.com)
24 plates with 88 siRNAs each
3 plates with 15 siRNAs each
See accompanying CD for sequence information and additional siRNA details.
Standard
Purity:
Annealed
Format:
Powder
Appearance:
Store at or below 鈥?20掳C. Do not store in a frost-free freezer. (Dried oligonucleotides are shipped at
Storage:
ambient temperature.)
1 year, when stored at or below 鈥?20掳C.
Shelf Life:
QUALITY CONTROL
The mass of a sample of each single-stranded RNA oligonucleotide is analyzed using MALDI-TOF mass
Identity:
spectrometry and compared to the calculated mass.
A sample of the annealed siRNA is analyzed by nondenaturing gel electrophoresis.
Annealing:
USER INFORMATION
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Silencer Human Kinase siRNA Library V3 targets 719 human kinases with three individual siRNAs per
Product Description:
gene. These chemically synthesized siRNAs are 21-mers comprised of 19 bp dsRNA molecules with
dinucleotide 3' DNA overhangs. The sequences of the siRNAs (provided in the accompanying CD) were
designed using a proprietary siRNA design algorithm that results in highly efficacious and specific siRNAs.
For more information about the Silencer Pre-designed siRNAs that make up this library, see
www.ambion.com/siRNA.
RNA oligonucleotides are susceptible to degradation by exogenous ribonucleases introduced during
Handling Instructions:
handling. Wear gloves when handling this product. Use RNase-free reagents, tubes, and barrier pipette tips.
Upon receipt, your siRNAs may be safely stored in a non-frost-free freezer at or below 鈥?20掳C (dried
oligonucleotides are shipped at ambient temperature).
Resuspension of siRNAs
Centrifuge each plate at low speed (maximum RCF 4,000 X g) to collect the contents at the bottom of the
wells before removing the seal in step 1.
Important: Perform this process under a tissue culture hood.
1. Remove seal carefully.
2. Add 50 碌L nuclease-free, sterile, water using a multichannel pipette and sterile tips (adding 50 碌L
water to 0.25 nmol siRNA will make a 5 碌M stock*).
3. Gently pipette up and down 5 times to resuspend.
4. (Optional) Aliquot the siRNAs into one or more daughter plates, to limit the number of freeze-thaw
cycles to which the siRNAs are subjected.
5. Place a new sterile seal (such as Axygen PCR-AS-200) on the plate before storing.
6. Store at 鈥?80掳C until ready to use.
* An online calculator for suspension of dried oligonucleotides is available at
www.ambion.com/techlib/append/oligo_dilution.html
Ambion, Inc. 鈥? An Applied Biosystems Business 鈥? +1 800-888-8804 鈥? www.ambion.com 鈥? e-mail: techserv@ambion.com
Cat #AM80010V3 Page 1 of 3
Lot #AMO07XDS
Silencer庐 Human Kinase siRNA Library Cat #AM80010V3
Applications: Transfecting siRNAs Into Mammalian Cells
The efficiency with which mammalian cells are transfected with siRNA will vary according to cell type and the
transfection agent used. This means that the optimal concentration used for transfections should be
determined empirically. We have found that siRNAs typically work best when present in cell culture medium
at 10鈥?50 nM; however, a more extensive concentration range from 1鈥?100 nM can be analyzed in
optimization experiments.
General Transfection Starting Points for Mammalian Cells
Plate Format 384 wells 96 wells 24 wells 12 wells
a
0.3 碌L 0.3鈥?1.0 碌L 1鈥?3 碌L 2鈥?4 碌L
Transfection Agent
b
2.1鈥?3 pmol 3 pmol 15 pmol 30 pmol
siRNA
c
4000鈥?8000 cells/well 6000 cells/well 40,000 cells/well 80,000 cells/well
Cell Density
70鈥?100 碌L 100 碌L 0.5 mL 1.0 mL
Final Volume per Well
a
Refer to the instructions provided with your transfection agent for the recommended volume.
b
The siRNA amount shown results in a final siRNA concentration of 30 nM. The amount of siRNA required for maximal gene
silencing will vary from cell type to cell type. For a 96-well plate, and a 100 碌L final transfection volume, 3 pmol of a 5 碌M siRNA
solution is 0.6 碌L. Robotic pipettors may require volumes of 2鈥?5 碌L for accurate pipetting. To increase pipetting volumes and
accuracy, we recommend preparing a plate with a dilution of your stock siRNA when preparing transfection complexes.
c
Optimal cell density will vary from cell type to cell type, depending on cell size and growth characteristics. In general, 30鈥?70%
confluency is recommended.
Transfection Optimization
Optimizing transfection efficiency is crucial for maximizing gene silencing while minimizing cytotoxicity.
Optimal transfection efficiencies are achieved by identifying an effective transfection agent for each cell type
and by adjusting (in order of importance):
鈥? Amount of transfection agent
鈥? Amount of siRNA
鈥? Cell density at the time of transfection
鈥? Order of transfection (pre-plating cells or plating cells/transfecting in tandem)
鈥? Length of exposure of cells to transfection agent/siRNA complexes
Most protocols recommend maintaining mammalian cells in the medium used for transfection; this avoids
diluting or removing the siRNAs from the cells by adding medium or washing the cells with new medium too
soon after transfection. We have found that cells typically exhibit greater viability when existing medium is
replaced by fresh medium 24 hours after transfection. Replacing medium after 24 hours generally does not
change the activity of the transfected siRNAs.
Once the conditions for maximal gene silencing are determined, they should be kept constant from
experiment to experiment for a given cell type. Include controls in all plates for each experiment to ensure
consistency.
For additional information about siRNA transfection, including transfection conditions for many cell types and
optimization protocols, see the Ambion siRNA Delivery Resource at:
www.ambion.com/techlib/resources/delivery
RELATED PRODUCTS
庐
Silencer Control siRNAs
Cat #Various (see www.ambion.com/siRNA)
Validated, nontargeting siRNAs (negative controls) and siRNAs targeting genes such as GAPDH, 尾-actin,
and GFP (positive controls).
siPORT鈩? NeoFX鈩? Transfection Agent
Cat #AM4510 and AM4511
A versatile lipid-based agent for efficient and reproducible transfection of adherent cells while subculturing,
without increased cytotoxicity.
庐
TaqMan Gene Expression Assays
www.allgenes.com
A comprehensive collection of over 700,000 probe and primer sets for quantitative gene expression analysis
using real-time PCR.
Ambion, Inc. 鈥? An Applied Biosystems Business 鈥? +1 800-888-8804 鈥? www.ambion.com 鈥? e-mail: techserv@ambion.com
Cat #AM80010V3 Page 2 of 3
Lot #AMO07XDS
Silencer庐 Human Kinase siRNA Library Cat #AM80010V3
OTHER INFORMATION
Material Safety Data Sheets (MSDSs) can be printed or downloaded from product-specific links on our website at the
Material Safety Data
following address: www.ambion.com/techlib/msds. Alternatively, e-mail your request to
Sheets:
MSDS_Inquiry_CCRM@appliedbiosystems.com. Specify the catalog or part number(s) of the product(s), and we will e-mail
the associated MSDSs unless you specify a preference for fax delivery. For customers without access to the internet or fax,
our technical service department can fulfill MSDS requests placed by telephone or postal mail. (Requests for postal delivery
require 1鈥?2 weeks for processing.).
For research use only. Not for use in diagnostic procedures.
Warranty and Liability:
Applied Biosystems is committed to delivering superior product quality and performance, supported by industry-leading global
service and technical support teams. Warranty information for the accompanying consumable product is available at
www.ambion.com/info/warranty in "Limited Warranty for Consumables," which is subject to the exclusions, conditions,
exceptions, and limitations set forth under the caption 鈥淓XCLUSIONS, CONDITIONS, EXCEPTIONS, AND LIMITATIONS鈥? in
the full warranty statement. Please contact Applied Biosystems if you have any questions about our warranties or would like
information about post-warranty support.
Applied Biosystems, AB (Design), Ambion, and Silencer are registered trademarks and siPORT and NeoFX are trademarks
Trademarks, Patents
of Applera Corporation or its subsidiaries in the US and/or certain other countries. TaqMan is a registered trademark of
and Licensing:
Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners.
NOTICE TO PURCHASER:
This product is licensed under European Patents 1144623, 1214945 and foreign equivalents from Alnylam Pharmaceuticals,
Inc., Cambridge, USA and is provided only for use in academic and commercial research including in vitro and in vivo
identification and validation of drug targets (but excluding the evaluation or characterization of this product as the potential
basis for a siRNA-based drug) and not for any other commercial purposes. Information about licenses for commercial use
(including discovery and development of siRNA-based drugs) is available from Alnylam Pharmaceuticals, Inc., 300 Third
Street, Cambridge, MA 02142, USA.
Ambion siRNA products are manufactured under license from the Massachusetts Institute of Technology to U.S. Patent
Nos. 7,056,704 and 7,078,196 and pending counterparts. The purchaser of the product may use it for research use only.
Purchase of this product gives the user rights under U.S. Patent No. 6,506,559, and its foreign equivalents, to use this
product for life science research, not for use in humans or clinical diagnostics.
漏 2007 Ambion, Inc. All rights reserved. 4393116A
Ambion, Inc. 鈥? An Applied Biosystems Business 鈥? +1 800-888-8804 鈥? www.ambion.com 鈥? e-mail: techserv@ambion.com
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