siRNA plasmid, pKD-FAK-v4

We recommend that you start with 100nM and test various concentrations between 1nM and 200nM to find the optimal concentration for your specific experimental conditions.

Dharmacon and Upstate have paired together reagents into a kit, which offer you value and convenience. There is a cost savings by buying the kit when compared to buying the kit components individually. Additionally, in most cases, Upstate antibodies are not offered with positive control cell lysates when purchased separately.

siRNA SMART pool® are a mixture of four individual siRNAs that have been designed with Dharmacon's proprietary algorithms, SMARTselection and SMARTpool. SMARTselection uses 34 criteria to eliminate the non-functional siRNAs. Then the SMARTpool technology uses a sophisticated algorithm to combine 4 or more SMART selected siRNA duplexes in a single pool, resulting in even greater probability that the siRNA pool reagent will reduce mRNA to low levels as compared to using a randomly designed single siRNA designed against the same gene.

The half-life of some proteins is particularly long. The siRNA SMART pool® mediated decrease in an mRNA may not be indicative of its protein level decrease if the gene product being targeted is very stable. Although we guarantee that the siRNA SMART pool® will reduce mRNA levels by at least 50%, we cannot guarantee that the level of a protein will decrease by a proportional level due to the variance in protein stability and cell systems.

We recommend using the siSTARTER kit to pilot your siRNA/siAB experiments. With this kit, you will be able to determine the efficacy of your transfections by knocking-down the expression of an internal control protein that has been well characterized to respond to siRNA-mediated knock-down: Lamin A/C. Additionally, you may use a host of control siRNA's offered by Dharmacon to control and optimize your experiments. Refer to www.dharmacon.com or call Dharmacon at 800-235-9880.

We recommend to start looking for a decrease 48 hours after transfection of the siRNA SMART pool®. However, the maximal decrease may be reached later. It is advisable to set up a time course experiment to identify optimal conditions. The cell system being used and the half-life of the specific protein being tested will determine when the maximal reduction is reached. The half-life of some proteins is particularly long and the level of its mRNA decrease may not be indicative of its protein level decrease.

We recommend to start looking for a decrease 24 hours after transfection of the siRNA SMART pool. However, the maximal decrease may be reached later. It is advisable to set up a time course experiment to identify optimal conditions. The cell system being used and the half-life of the specific mRNA being tested will determine when the maximal reduction is reached.

The pKD's are guaranteed to generate 70% knockdown in HeLa cells. No such guarantee is given for Jurkats due to the fact that they are notoriously difficult to transfect. If you must use Jurkats, we recommend that you try Jurkat specific electroporation protocols. There is some data that suggests that higher transfection efficiencies can be achieved when utilizing specific protocols. Additionally, a selectable marker for stable generation would circumvent the need for higher transfection efficiencies. While we don't have a stable plasmid version available, there are restriction sites that can be used downstream of the shRNA where a selectable marker can be cloned.

This plasmid contains Ampicillian as the bacterial selection marker. However, it does not contain a stable transfection marker and therefore cannot be stably transfected.

It is known that double-stranded RNA (dsRNA) in mammalian cells is able to trigger a strong physiological response that results in a global, translational inhibition and mRNA degradation. dsRNA can initiate interferon synthesis whereby PKR and 2', 5'-oligoadenylate synthase become activated. Activated PKR phosphorylates translational initiation factors, which causes the cell's translational machinery to stall. Activated 2', 5'-oligoadenylate synthase results in the activation of ribonuclease L that causes cellular mRNA degradation. Together, this results in a general, non-specific, dsRNA-mediated silencing of gene expression. However, using dsRNA, that is shorter than 30bp in length, allows RNAi to occur without activating the interferon response. The short length of the dsRNA allows the interferon response to be bypassed. This prevents the non-specific knockdown of gene expression. The dsRNA molecules in the siRNA SMART pools are siRNA duplexes shorter than 30bp, and therefore will not active the interferon-mediated PKR response. Please see reference: Elbashir, S. M., et al. (2001) Nature 411, 494-498.

Yes, please inquire for pricing and availability. However, we do not guarantee the performance of individual duplexes.

Currently you can receive partial (14/19) or full sequence information (14/19) and 1nmole of each individual duplex at an additional cost with terms and conditions.

Dharmacon's pooling algorithm is proprietary information.

The 1X Universal Buffer composition is 20mM KCl, 6mM HEPES (pH 7.5), 0.2mM MgCl2.

No, the siRNA SMART pools have been designed against the homologous regions of the different isoforms of the same gene.

2'-ACE protected RNA, either duplexed or single stranded is completely resistant to nucleases by virtue of the protecting group. A deprotected duplex is significantly more resistant to nucleases than deprotected single strands. However, maintaining sterile, RNAse free conditions is always recommended as a precaution.

The duplexes that comprise an siRNA SMARTpools are processed as Option A4 duplexes (deprotected/desalted; annealed). The duplexes all have "UU" overhangs and a 5' phosphate on the antisense strand.

We recommend to start looking for a decrease 24 hours after transfection of the siRNA SMART pool®. However, the maximal decrease may be reached later. It is advisable to set up a time course experiment to identify optimal conditions. The cell system being used and the half-life of the specific mRNA being tested will determine when the maximal reduction is reached.

The siRNA SMART pool® reagent provided in this kit has been designed to target human sequences. The mRNA sequence that is targeted is indicated by the GenBank accession number. We guarantee the efficacy when used in human cell lines. However, we have not tested this specific duplex effectiveness in other species.