Line Silence RNAi Complete Expression Kit

LineSilenceTM RNAi Complete
Expression Kit
L ineSilenceTM RNAi cassettes (pro-

tected under US patent 7,294,504
and additional patent pending) use
Box 1 | Product Summary
engineered human U6 polymerase III Catalogue Numbers ABP-RI-LS01040

promoter and modified terminator for ABP-RI-LS01080
high level, precise siRNA expression Components Template (1 ng/μl)
inside mammalian cells. Cloning is not Upstream primer (20 μM)
necessary. It takes only a few hours p53-S (20 μM) (for generating sense siRNA for
to generate RNAi reagents ready for p53)
transfection. It is particularly suitable p53-A (20 μM) (for generating antisense siRNA
for screening for effective RNAi tar- for p53)
gets with convenience at low cost. If Allele-in-one PCR Mix
desired, these cassettes can be easily AvantGene
cloned into any vector, e.g. plasmid or DNA Diluent
virus, for amplification or other special p53-5’ PCR primer (20 μM)
purposes. p53-3’ PCR primer (20 μM)
Actin-5’ PCR primer (20 μM)
Features Actin-3’ PCR primer (20 μM)
Storage Store the AvantGene at +4°C. All other rea-
T he LineSilencetm Complete
RNAi kit is suitable for RNAi
experiments in tissue culture cells.
Stability
gents to be stored at -20°C.
All components are stable for 6 months when
stored properly.
Each batch of reagents is vigorously
tested for consistency and stability,
and offer the following features:
- Efficient RNA interference
- Suitable for high throughput RNAi
target screening cassettes that have been tested Design of Inserts
to significantly reduce p53 mRNA
- Amenable to conversion to plasmid
levels are also included as positive
construct by simple cloning
controls. Allele-In-One PCR MasterMix Choose a target region that is A2N19
- Lower cost compared to synthetic is provided for high quality and (sense sequence of the target RNA).
siRNA convenient PCR reactions to To generate antisense siRNA
generate the cassette DNA. The transcript, the following primer is
PCR protocol is optimized to yield synthesized:
Materials Not Suppplied sufficient LineSilence products
with virtually all primers tested. 5’ caaaaac tgtaaaaa
with the Kit
The Complete Kit also provides Terminator
AvantGene™ transfection reagent, N19 gg tgt ttc gtc ctt tcc aca aga 3’
-PCR purification system which is suitable for most mammalian Template matching region
-Downstream gene-specific primers cells with exceptional efficiency.
-RNA purification system and reverse To generate sense siRNA transcript,
transcription enzyme Note: This product may be protected the above N19 sequences is replaced
under US patent 7,294,504 and additional with its reverse/complementary
pending patents. Purchasing of this prod-
sequence to
uct grants the rights of use. Commercial
Reagents Provided with user may be required to obtain further
the Kit license from third parties in order to use N’19:
certain RNA interference related technolo- 5’caaaaac tgtaaaaa
gies. Terminator
The kit provides enough PCR N’19 gg tgt ttc gtc ctt tcc aca aga 3’
T-PCR primers for detecting p53 and Template matching region
template and upstream primer to
actin mRNA levels are also included
synthesize up to 100μg of linear DNA
in the Complete Kit. A successful
expression cassettes (correspond-
p53 RNAi experiment is expected to
ing to about 200 transfections on a
result in reduced p53 mRNA levels
24-well plate). Gene-specific down-
while not affecting actin mRNA levels.
stream PCR primers for each RNAi
RT-PCR with p53 primers should
target must be designed according to
generate a band of 496 bp; RT-PCR
the guidelines listed below. Primers
with actin primers results in a band of
for generating p53-specific RNAi
587bp.
Allele Biotech-Introducing Cost Effectiveness to Research

Protocols
PCR Protocol Transfection
Using Allele-In-One MasterMix: Cells are prepared and transfected generally as you would
Component Volume Final Concentra- with a typocal expression plasmid transfection. Most
tion commercial transfection reagents may be used with the
LineSilence cassette reagents. Although using AvantGene
Template DNA (1ng/µl) 1 µl 20 pg/µl transfection reagent is recommended, in many cases the
Upstream Primer (20µM) 1.5 µl 0.6 µM choice os transfection reagent should depend on cells to
Downstream Gene-Specific 1.5 µl 0.6 µM be used. Use 0.5 μg of each sense and antisense Line-
Primer (20µM) Silence cassettes per well of a 24-well plate as a starting
point.
Distilled Water 21 µl
All -In-One MasterMix
ele
25 µl Using AvantGene
The following procedure is suggested as a starting point * All Volumes are for each well of 24-well plate:
when using Taq polymerase:
Component Volume Final Concentra- 1. Plate cells approximately 24 hours prior to transfection
tion at a cell density of 20-40% confluence in complete medium
(with serum and antibiotics if required).
Template DNA (1ng/µl) 1 µl 20 pg/µl
10X PCR Buffer 5 µl 1X 2. Mix 2.5 μl AvantGene reagent to 10 μl serum free, antibi-
otics-free medium, incubate 5-10 min at room temperature.
10mM dNTP Mix 1 µl 0.2 mM
Upstream Primer (20µM) 1.5 µl 0.6 µM 3. Add 12.5 μl DNA Diluent to 0.5 μg DNA, incubate 1-5
Downstream Gene-Specific 1.5 µl 0.6 µM min at room temperature. Do not incubate longer than 5
Primer (20µM) min.
Taq DNA Polymerase 5 unit
4. While waiting, change cell medium to 200 μl serum-free
Distilled Water 50 µl and antibiotics-free medium.
Perform 30-40 cycles of PCR amplification (2-step) as fol- 5. Mix diluted transfection reagent from Step 2 with DNA
lows: solution from Step 3, incubate at room temperature for 5-10
Denature 98°C for 30 sec min.
Anneal and Extend 72°C for 1 min 30 sec
6. Add the transfection mixture from step 5 drop-wise to
cells.
Normally and 200 μl reaction is needed to produce enough
DNA (5-10 μg after purification) for 10 to 20 transfections 7. After 2-4 hours incubation under appropriate condi-
on a 24-well plate. It would be beneficial to purify the PCR tions in an incubator, add 250 μl serum-containing normal
products with a PCR DNA purification kit. medium.
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
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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Line Silence RNAi Complete Expression Kit

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Available Formats

LineSilenceTM RNAi Complete

L ineSilenceTM RNAi cassettes (pro-

engineered human U6 polymerase III Catalogue Numbers ABP-RI-LS01040

Allele Biotech-Introducing Cost Effectiveness to Research

F or Research Use Only. Not for

Allele Biotech-Introducing Cost Effectiveness to Research

Allele Biotech-Introducing Cost Effectiveness to Research

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