SilenCircleTM Complete RNAi

Expression Kit

S ilenCircleTM RNAi system (pro-

tected under US patent 7,294,504
and additional patents pending) is
Box 1 | Product Summary

a plasmid-based RNA interference Catalogue Numbers ABP-RI-SC01010

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-to-
ligate 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 knock-
down expression of both endogenous
and exogenous genes.
Features
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)
Storage Store the AvantGene at +4°C. All other rea-
gents to be stored at -20°C.
Stability All components are stable for 6 months when
stored properly.

T he SilenCircletm Complete RNAi

kit is suitable for RNAi experi-
ments 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.
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.
F 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
Reagents Provided with
the Kit
T he kit provides enough reagents
to construct 10 RNAi expressing
plasmids, including Allele recom-
binant 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.
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 comple-
mentary 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/ comple-
mentary 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 prod-
uct 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 technolo-
gies.

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Protocols
Cloning Transfection
Oligo DNA annealing, ligation into the linearized vector, E. Cells are prepared and transfected generally as you
coli transformation, and plasmid DNA preparation may be would with a typical expression plasmid transfection. Most
performed according to standard protocols. Plasmid DNA commercial transfection reagents may be used with the
from positive clones will be cut only once by restriction SilenCircleTM plasmids. Although using AvantGene trans-
enzyme Stu I on the plasmid backbone (The Stu I site in fection reagent is recommended, in many cases the choice
the Polylinker should have been deleted from the pre-cut of transfection reagent should depend on cells to be used.
vector). A self-ligation plasmid (from the very few molecules Use 0.5 μg plasmid DNA per well of a 24-well plate as a
that escaped linearization of the vector) will yield two bands starting point.
of 1.1 kb and 3.5 kb, respectively. A sequencing primer is
also included in the kit for positive clone verification. Using AvantGene
Suggested Protocol for siRNA Insert Prepa- * All Volumes are for each well of 24-well plate:
ration
1. Plate cells approximately 24 hours prior to transfection
Top oligo 1μg at a cell density of 20-40% confluence in complete medium
(with serum and antibiotics if required).
Bottom Oligo 1μg
Annealing Buffer (10X) 2μl 2. Mix 2.5 μl AvantGene reagent to 10 μl serum free, antibi-
Distilled Water 20μl otics-free medium, incubate 5-10 min at room temperature.

Heat at 95°C for 10min, slowly cool down to room tempera- 3. Add 12.5 μl DNA Diluent to 0.5 μg DNA, incubate 1-5
ture. min at room temperature. Do not incubate longer than 5
min.
Suggested Protocol for DNA Ligation Using
Allele’s hcT4 DNA Ligase 4. While waiting, change cell medium to 200 μl serum-free
Pre-Cut Vector (ng/μl) 2μl and antibiotics-free medium.
Annealed insert 2μl 5. Mix diluted transfection reagent from Step 2 with DNA
Ligand buffer (10) 1μl solution from Step 3, incubate at room temperature for 5-10
T4 DNA ligase 0.5μl min.

Distilled water 4.5μl 6. Add the transfection mixture from step 5 drop-wise to
cells.
Incubate at room temperature for an hour or at 4-16°C
overnight. 7. After 2-4 hours incubation under appropriate condi-
tions in an incubator, add 250 μl serum-containing normal
medium.
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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