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SilenCircleTM Complete RNAi Expression Kit

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ilenCircleTM RNAi system (protected under US patent 7,294,504 and additional patents pending) is a plasmid-based RNA interference system that uses engineered human U6 RNApolymerase III promoter and modified terminator for high level small hairpin RNA (shRNA) or siRNA expression inside mammalian cells. The design enables precise start and end of an interfering RNA with the optimal 3’ overhanging nucleotides. The pre-cut linear vector is ready-toligate for construction of interfering RNA expression plasmid with nearly zero self-ligation. Bearing a neomycin resistant marker, it may be used for establishing siRNA-expressing stable cell lines. SilenCircle RNAi system has been successfully used to knockdown expression of both endogenous and exogenous genes.

Box 1 | Product Summary
Catalogue Numbers ABP-RI-SC01010 ABP-RI-SC01020 Components pre-cut pSIlenCircle Vector (10ng/µl) pSilenCircle sequencing Primer (20µM) p53-top (coding for p53 RNAi insert top strand) (20µM) p53-bot (coding for p53 RNAi insert bottom strand) (20µM) Annealing Buffer (10X) hcT4 DNA Ligase with 10X Buffer AvantGene DNA Diluent p53-5’ PCR Primer (20µM) p53-3’ PCR Primer (20µM) Actin-5’ PCR Primer (20µM) Actin-3’ PCR Primer (20µM) Store the AvantGene at +4°C. All other reagents to be stored at -20°C. All components are stable for 6 months when stored properly.

Storage Stability

Features

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he SilenCircletm Complete RNAi kit is suitable for RNAi experiments in tissue culture cells. Each batch of reagents is vigorously tested for consistency and stability, and offer the following features: - Efficient RNA interference - Convenient ready-to-ligate format - Almost no background ligation - Without introducing extra bases from restriction enzyme sites, generate precise shRNA, siRNA, or miRNA.

Reagents Provided with the Kit

Design of Inserts
siRNA may be produced from two pSilenCircle plasmids encoding either sense or antisense. Alternatively, shRNA or miRNA may be produced from a single plasmid (shown below). For each siRNA insert, two complementary synthetic DNA oligos are needed. Choose a target region that is A2N19 (sense sequence of the target RNA), design a linker sequence (e.g. 9 bases), use the following format: 5’ acacc N19 Linker N’19 t 3’ 3’ g N’19 rcLinker N19 aaaaa 5’ # “N’19” is reverse/complementary to “N19”; “rclinker” is reverse/ complementary to “linker”. # Oligos orders are typically entered from 5’ to 3’, i.e. aaaaa N19...
Note: This product may be protected under US patent 7,294,504 and additional pending patents. Purchasing of this product grants the rights of use. Commercial user may be required to obtain further license from third parties in order to use certain RNA interference related technologies.

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he kit provides enough reagents to construct 10 RNAi expressing plasmids, including Allele recombinant high concentration T4 DNA ligase (hcT4 DNA ligase). DNA oligos encoding each RNAi target sequence need to be designed according to the guidelines listed below. Oligos for generating p53-specific RNAi cassettes that have been tested to significantly reduce p53 mRNA levels are also included as positive control. The Complete Kit also provides AvantGeneTM transfection reagent, suitable for most mammalian cells with exceptional efficiency. RT-PCR primers for detecting p53 and actin mRNA levels are also included in the Complete Kit. A successful p53 RNAi experiment is expected to result in reduced p53 mRNA levels while not affecting actin mRNA levels. RT-PCR with p53 primers should generate a band of 496 bp; RT-PCR with actin primers results in a band of 587b.

Materials Not Suppplied with the Kit
-Target-specific insert DNA oligos -E. coli competent cells -Plasmid DNA purification system -RNA purification system and reverse transcription enzyme.
or Research Use Only. Not for Diagnostic or Therapeutic Use. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of Allele Biotech is strictly prohibited

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Protocols
Cloning
Oligo DNA annealing, ligation into the linearized vector, E. coli transformation, and plasmid DNA preparation may be performed according to standard protocols. Plasmid DNA from positive clones will be cut only once by restriction enzyme Stu I on the plasmid backbone (The Stu I site in the Polylinker should have been deleted from the pre-cut vector). A self-ligation plasmid (from the very few molecules that escaped linearization of the vector) will yield two bands of 1.1 kb and 3.5 kb, respectively. A sequencing primer is also included in the kit for positive clone verification.

Transfection
Cells are prepared and transfected generally as you would with a typical expression plasmid transfection. Most commercial transfection reagents may be used with the SilenCircleTM plasmids. Although using AvantGene transfection reagent is recommended, in many cases the choice of transfection reagent should depend on cells to be used. Use 0.5 μg plasmid DNA per well of a 24-well plate as a starting point.

Using AvantGene
* All Volumes are for each well of 24-well plate: 1. Plate cells approximately 24 hours prior to transfection at a cell density of 20-40% confluence in complete medium (with serum and antibiotics if required). 2. Mix 2.5 μl AvantGene reagent to 10 μl serum free, antibiotics-free medium, incubate 5-10 min at room temperature. 3. Add 12.5 μl DNA Diluent to 0.5 μg DNA, incubate 1-5 min at room temperature. Do not incubate longer than 5 min. 4. While waiting, change cell medium to 200 μl serum-free and antibiotics-free medium. 5. Mix diluted transfection reagent from Step 2 with DNA solution from Step 3, incubate at room temperature for 5-10 min. 6. Add the transfection mixture from step 5 drop-wise to cells. 7. After 2-4 hours incubation under appropriate conditions in an incubator, add 250 μl serum-containing normal medium.

Suggested Protocol for siRNA Insert Preparation
Top oligo Bottom Oligo Annealing Buffer (10X) Distilled Water 1μg 1μg 2μl 20μl

Heat at 95°C for 10min, slowly cool down to room temperature.

Suggested Protocol for DNA Ligation Using Allele’s hcT4 DNA Ligase
Pre-Cut Vector (ng/μl) Annealed insert Ligand buffer (10) T4 DNA ligase Distilled water 2μl 2μl 1μl 0.5μl 4.5μl

Incubate at room temperature for an hour or at 4-16°C overnight.

Method Overview

Website: www.allelebiotech.com Call: 1-800-991-RNAi/858-587-6645 (Pacific Time: 9:00AM~5:00PM) Email: oligo@allelebiotech.com

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