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File Name: ambion_com---sp_80140v3.asp
 Silencer庐 Phosphatase siRNA Library Product Information Sheet

Silencer Phosphatase siRNA Library
Product:

80140V3
Catalog #:

AMO0169W
Lot #:

0.25 nmol each siRNA
Amount:

801 unique siRNAs targeting each of 267 phosphatase genes
Content:
Total of 12 96-well plates*
9 plates with 80 siRNAs each
3 plates with 27 siRNAs each
See accompanying CD for sequence information and additional siRNA details.

*Axygen #PCR96FS; www.axygen.com

Standard
Purity:

Annealed
Format:

Powder
Appearance:

Store at 鈥?20掳C or 鈥?80藲C. Do not store in a frost-free freezer. (Dried oligonucleotides are shipped at ambient
Storage:
temperature.)

1 year, when stored at 鈥?20掳C or 鈥?80藲C.
Shelf Life:


QUALITY CONTROL

A MALDI-TOF mass spectrometer is used to identify the correct mass of the single-stranded RNA
Identity:
oligonucleotides.

Analytical HPLC of the final purified single-stranded RNA oligonucleotides is used to confirm >80% purity.
Purity:

The annealed siRNA is analyzed by nondenaturing PAGE.
Annealing:


USER INFORMATION

RNA oligonucleotides are susceptible to degradation by exogenous ribonucleases introduced during
General Handling
handling. As a precaution, your siRNAs should not be handled with ungloved hands. RNase-free reagents,
Instructions:
barrier pipette tips, and tubes should be used. Upon receipt, your siRNAs may be safely stored in a non-
frost-free freezer for 1 year at 鈥?20掳C or 鈥?80藲C (dried oligonucleotides are shipped at ambient temperature).

Centrifuge each plate at low speed to collect the contents at the bottom of the wells before removing the seal
Resuspension of
in step 1.
siRNAs:

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 on the plate before storing.
6. Store at 鈥?80掳C until ready to use.


The efficiency with which mammalian cells are transfected with siRNA will vary according to cell type and the
Transfecting siRNAs
transfection agent used. This means that the optimal concentration used for transfections should be
Into Mammalian Cells:
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.




80140V3_PISheet_0606v1 Page 1 of 3
Page 2 of 3

Silencer庐 Phosphatase siRNA Library Cat #80140V3

The following chart provides helpful starting points for transfection of siRNA into cultured mammalian cells.
General Transfection
Starting Points:

384 well 96 well 24 well 12 well
a
0.3 0.3鈥?1.0 1鈥?3 2鈥?4
Reagent (碌l)
b
2.1鈥?3 3 15 30
siRNA (pmol)
c
4000鈥?8000 6000 40,000 80,000
Cell density (cells/well)

0.07鈥?0.1 0.1 0.5 1.0
Final volume per well (ml)

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.

Optimizing transfection efficiency is crucial for maximizing gene silencing while minimizing cytoxicity.
Transfection
Optimal transfection efficiencies are achieved by identifying an effective transfection agent for each cell type
Optimization:
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 Ambion's siRNA Delivery Resource at:
http://www.ambion.com/techlib/resources/delivery

RELATED PRODUCTS

Silencer Control siRNAs
Cat #Various (see www.ambion.com/siRNA)
Validated, non-targeting siRNAs (negative controls) and siRNAs targeting genes such as GAPDH, 尾-actin,
and GFP (positive controls).

siPORT鈩? NeoFX鈩? Transfection Agent
Cat #4510 and 4511
A versatile lipid-based agent for efficient and reproducible transfection of adherent cells while subculturing,
without increased cytotoxicity.

OTHER INFORMATION
Material Safety Data Sheets (MSDSs) can be printed or downloaded from our website by going to the following address and
Material Safety Data
clicking on the link for the item: www.ambion.com/techlib/msds.
Sheets:
Alternatively, e-mail us at MSDS@ambion.com to request MSDSs by e-mail, fax, or ground mail. Specify the Ambion
catalog number of the item(s) for which you want MSDSs and whether you want to receive the information by e-mail, fax, or
ground mail. Be sure to include your fax number or mailing address as appropriate. If the mode of receipt is not specified,
we will e-mail the MSDSs. Customers without internet access can contact our Technical Service Department by telephone,
fax, or ground mail to request MSDSs.

This product is licensed under European Patents 1144623, 1214945 and foreign equivalents from Alnylam Pharmaceuticals,
Patents and
Inc., Cambridge, USA and is provided only for use in academic and commercial research including in vitro and in vivo
Licensing:
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

80140V3_PISheet_0606v1

Ambion, Inc. (Headquarters) USA: 800-888-8804 鈥? Can: 800-445-1161 鈥? Intl: +1-512-651-0200 鈥? www.ambion.com 鈥? e-mail: techserv@ambion.com
Ambion (Europe) Ltd. 鈥? tel: +44(0)1480-373-020 鈥? fax +44(0)1480-373-010 鈥? www.ambion.com 鈥? e-mail: eurotech@ambion.com
Ambion K.K. (Japan) 鈥? tel: +81(0)3-5638-2181 鈥? fax: +81(0)3-5638-2182 鈥? www.ambion.co.jp 鈥? e-mail: jtech@ambion.com
www.ambion.com/techlib/spec/sp_80140v3.pdf
Page 3 of 3

Silencer庐 Phosphatase siRNA Library Cat #80140V3

(including discovery and development of siRNA-based drugs) is available from Alnylam Pharmaceuticals, Inc., 300 Third
Street, Cambridge, MA 02142, USA.

Ambion has been granted rights by the Massachusetts Institute of Technology to US Patent Applications 60/265232,
09/821832 and PCT/US01/10188, RNA Sequence-Specific Mediators of RNA Interference. The purchaser of the product
may use it for research use only.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Warranty and Liability:
Ambion is committed to providing the highest quality reagents at competitive prices. Ambion warrants that the products
meet or exceed the performance standards described in the product specification sheets. If you are not completely satisfied
with any product, our policy is to replace the product or credit the full purchase price and delivery charge. No other
warranties of any kind, expressed or implied, are provided by Ambion. Ambion's liability shall not exceed the purchase price
of the product. Ambion shall have no liability for direct, indirect, consequential or incidental damages arising from the use,
results of use, or inability to use its products.




80140V3_PISheet_0606v1

Ambion, Inc. (Headquarters) USA: 800-888-8804 鈥? Can: 800-445-1161 鈥? Intl: +1-512-651-0200 鈥? www.ambion.com 鈥? e-mail: techserv@ambion.com
Ambion (Europe) Ltd. 鈥? tel: +44(0)1480-373-020 鈥? fax +44(0)1480-373-010 鈥? www.ambion.com 鈥? e-mail: eurotech@ambion.com
Ambion K.K. (Japan) 鈥? tel: +81(0)3-5638-2181 鈥? fax: +81(0)3-5638-2182 鈥? www.ambion.co.jp 鈥? e-mail: jtech@ambion.com
www.ambion.com/techlib/spec/sp_80140v3.pdf

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