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ShortCut siRNA Mixes >
Akt1/2/3 Kinases ShortCut® siRNA Mix |
 | | | | Akt1/2/3 Kinases ShortCut® siRNA Mix | | | |
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Prices are in US dollars and valid only for US orders.
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Description:
A heterogeneous mixture of 21-23 bp short interfering RNAs (siRNA) that induces effective silencing (RNAi) of human Akt1/2/3 Kinases in mammalian cell lines.
The serine-threonine protein kinases Akt1, Akt2 and Akt3 are critical mediators of growth factor-induced cell survival and are capable of phosphorylating several known proteins (1,2,3).
Recommended Usage Concentration: 5 to 20 nM
Figure 1: 15 ng (1 pmol) of ShortCut siRNA mix is run along with a synthetic 21 and 25 bp siRNA markers (NEB #N2101) on a 20% TBE polyacrylamide gel electrophoresis.
Figure 2: HeLa cells were transfected with 15 nM Akt1/2/3 ShortCut siRNA Mix (+) or 15 nM Lit28i Polylinker siRNA Mix (NEB #N2014) as a control (C) using TransPass R1 Transfection Reagent (NEB #M2551). Effective silencing of Akt1/2/3 is achieved at a low siRNA concentration 48 hours post-transfection. Akt1/2/3 was detected in a Western blot of cell lysates using Cell Signaling Technology (CST #9272) Antibody and actin loading control was detected using (Sigma A-2066) Antibody.
Background:
ShortCut® siRNA Mixes are highly potent, validated silencing mixtures generated by NEB's proprietary ShortCut RNase III technology. The section of mRNA sequence that each mixture targets has been selected to ensure no significant sequence identity to other genes. Each mixture is a unique combination of many individual siRNAs that work collectively to knock down the target gene. As a result, the mixtures can be used at extremely low concentration (typically 1-20 nM) which minimizes the potential for off-target effects.
Source:
A 1038 bpDNA template derived from mouse Akt1 cDNA (accession number NM_009652, coordinates 284?1322) was used to generate dsRNA which was processed by ShortCut RNase III to produce the siRNA Mix.
The mouse Akt1 DNA template has a 91% sequencing identity to human Akt1, a 79% sequence identity to human Akt2 and a 74% sequence identity to human Akt3. The siRNA Mix is effective against all three Akt proteins.
Reaction & Storage Conditions
Concentration:
10 μM
Storage Conditions:
20 mM KCl
0.5 mM EDTA
10 mM Na-HEPES
pH 7.0 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- It is important to maintain healthy cells. Some cell lines become more sensitive to transfection agents after a large number of passages. It is advisable to use cells subjected to a similar number of passages to ensure reproducible transfection results in different experiments.
- It is recommended that control transfections be performed by varying cell confluence and using different amounts of TransPass Transfection Reagent. In general, low cell density or too much transfection reagent increase the risk of cell toxicity. The medium may be replaced 24 hours after transfection with fresh complete medium to increase cell viability.
In order to easily estimate the efficiency of transfection of particular cell lines use the Fluorescein-siRNA Transfection Control (NEB #N2100S). - TransPass R1 Transfection Reagent has been used to successfully transfect siRNAs in many cell lines including: A549, C6, CHO, COS-7, HEK293, NIH 3T3, HepG2, HCT116, HeLa, Jurkat, MCF-7, U2OS, 3T3-L1 preadipocytes.
- Transfection of siRNA Mixtures: The transfection of siRNA must be optimal in order to obtain maximum silencing of a target gene. Optimizing the following parameters may be necessary in order to maximize the transfection efficiency for a particular cell line: the cell density at the time of transfection, the amount of transfection reagent, the amount of siRNA and the culture incubation time before analysis.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Purified by ion exchange chromatography free of contaminating large molecular weight dsRNA, ssRNA, DNA, bacteria and protein.
References
- Franke, T.F. (1997) Cell, 88, 435-437.
- Burgering, B.M. and Coffer, P.J. (1995) Nature, 376, 599-602.
- Franke, T.F. et al. (1995) Cell, 81, 727-736.
Companion Products
Fluorescein-siRNA Transfection Control
Lit28i Polylinker ShortCut® siRNA Mix
TransPass™ R1 Transfection Reagent
TransPass™ R2 Transfection Reagent
Legal
Licenses/Patents/Disclaimers:
Notice to Buyer/User: Commercial use of ShortCut® siRNA Mixes may require a license from New England Biolabs, Inc. Use of ShortCut® siRNA Mixes for RNA interference may require a license from the Carnegie Institution of Washington. For further information, contact the Director of Administration and Finance at the Carnegie Institute of Washington at 1530 P Street N.W., Washington, DC 20005-1910. Tel: 202-939-1118.
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