SilenCircleTM mTFP1 plus

Basic RNAi Expression Kit

S ilenCircleTM RNAi system (pro-
tected 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 op-
timal 3’ overhanging nucleotides. The
pre-cut linear vector is ready-to-ligate
for construction of interfering RNA ex-
pression plasmid with nearly zero self-
ligation. Bearing a neomycin resistant
marker, it may be used for establish-
ing siRNA-expressing stable cell lines.
It alslo contains an mTFP1 marker for
FP analysis. SilenCircle RNAi system
has been successfully used to knock-
down expression of both endogenous
and exogenous genes.
Box 1 | Product Summary
Catalogue Numbers ABP-RI-SC04010 (10 rxns)
ABP-RI-SC04020 (20 rxns)
Components Pre-Cut pSIlenCircle Vector also expressing
mTFP1 (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)
Storage All reagents to be stored at -20°C.
Stability All components are stable for 6 months when
stored properly.
Reagents Provided with
the Kit Design of Inserts
siRNA may be produced from two
pSilenCircle plasmids encoding either

T he kit provides enough reagents sense or antisense. Alternatively,

Features
T he SilenCircletm mTFP1 plus
Basic 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.
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.
-Annealing buffer (10X)
-hcT4 DNA ligase with 10X buffer
-AvantGene Transfection Reagent
-DNA dilutent
-5’ and 3’ p53 PCR primers
-5’ and 3’ Actin PCR primers
to construct 10 or 20 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 Basic Kit also provides 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.
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
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 trans-
fection 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.

Suggested Protocol for siRNA Insert Prepa-

F or Research Use Only. Not for
Diagnostic or Therapeutic Use.
Purchase does not include or carry
ration any right to resell or transfer this
product either as a stand-alone
product or as a component of another
Top oligo 1μg product. Any use of this product other
Bottom Oligo 1μg than the permitted use without the
express written authorization of Allele
Annealing Buffer (10X) 2μl
Biotech is strictly prohibited
Distilled Water 20μl

Heat at 95°C for 10min, slowly cool down to room tempera-

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

Method Overview

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