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LineSilenceTM RNAi Complete

Mouse Kit

L ineSilence™ RNAi
(protected under US patent
7,294,504 and additional patent
cassettes
Box 1 | Product Summary

pending) use engineered mouse U6 Catalogue Numbers ABP-RI-LS02040 (40 rxns)


polymerase III promoter and modified ABP-RI-LS02080 (80 rxns)
terminator for high level, precise siRNA Components Template (1 ng/μl)
expression inside mammalian cells. Upstream primer (20 μM)
Cloning is not necessary. It takes only p53-S (20 μM) (for generating sense siRNA for
a few hours to generate RNAi reagents p53)
ready for transfection. It is particularly p53-A (20 μM) (for generating antisense siRNA
suitable for screening for effective for p53)
RNAi targets with convenience at low Allele-in-one PCR Mix
cost. If desired, these cassettes can AvantGene
be easily cloned into any vector, e.g. DNA Diluent
plasmid or virus, for amplification or p53-5’ PCR primer (20 μM)
other special 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 Mouse


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
significantly reduce p53 mRNA levels
- Suitable for high throughput RNAi are also included as positive controls.
target screening Allele-In-One PCR MasterMix
Design of Inserts
- Amenable to conversion to plasmid is provided for high quality and Choose a target region that is A2N19
construct by simple cloning convenient PCR reactions to generate (sense sequence of the target RNA).
- Lower cost compared to synthetic the cassette DNA. The PCR protocol
siRNA is optimized to yield sufficient To generate antisense siRNA
LineSilence products with virtually all transcript, the following primer is
primers tested. The Complete Kit also synthesized:
Materials Not Suppplied provides AvantGeneTM transfection
reagent, which is suitable for most 5’ caaaaac tgtaaaaa →
with the Kit mammalian cells with exceptional Terminator
efficiency. →N19 aa aca agg ctt ttc tcc aag gga 3’
Template matching region
-PCR purification system
-Downstream gene-specific primers Note: This product may be protected
under US patent 7,294,504 and additional To generate sense siRNA transcript,
-RNA purification system and reverse
pending patents. Purchasing of this product the above N19 sequences is replaced
transcription enzyme
grants the rights of use. Commercial user with its reverse/complementary
may be required to obtain further license sequence to N’19:
Reagents Provided with from third parties in order to use certain
5’caaaaac tgtaaaaa →
RNA interference related technologies.
the Kit Terminator
T-PCR primers for detecting p53 and →N’19 aa aca agg ctt ttc tcc aag gga 3’
The kit provides enough PCR template
actin mRNA levels are also included Template matching region
and upstream primer to synthesize up
to 100 μg of linear DNA expression in the Complete Kit. A successful p53
cassettes (corresponding to about RNAi experiment is expected to result
200 transfections on a 24-well plate). in reduced p53 mRNA levelswhile not
Genespecific downstream PCR affecting actin mRNA levels. RT-PCR
primers for each RNAi target must be with p53 primers should generate a
designed according to the guidelines band of 466 bp; RT-PCR with actin
listed below. Primers for generating primers results in a band of 540 bp.
p53-specific RNAi cassettes that have
been tested to

Allele Biotech-Introducing Cost Effectiveness to Research


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

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

Allele Biotech-Introducing Cost Effectiveness to Research


Method Overview

Allele Biotech-Introducing Cost Effectiveness to Research