Download Forever 100bp Ladder Personalizer

Forever 100bp
Ladder Personalizer
User Manual
Version 1.0
Published September 20, 2003
Catalog No. FBLP-2003
Storage Conditions: -20℃
For Research Use Only
Product Warranty and Liability
Seegene warrants the performance of all products as described when used according
to instruction. Any problem incurred for any reason, other than misuse, should be
reported to Seegene immediately. This warranty limits our liability to replacement of the
products.
Safety Warning and Precautions
This product is limited for research use only, not recommended or intended for
diagnosis of disease in humans or animals. Do not use internally or externally in
humans nor animals.
Ordering Information and Technical Services
Seegene USA
P.O. Box N
Del Mar, CA 92014-0376
USA
Tel : + 858-509-3044
Fax: + 858-509-7596
E-mail: [email protected]
URL: www.see-gene.com
Seegene, Inc.
142-21 Samsung-dong, Kangnam-gu
Seoul, 135-090
Korea
Tel: + 82 2-566-9830
Fax: +82 2-566-9831
E-mail: [email protected]
URL: www.see-gene.com
The PCR process is covered by patents owned by Hoffman-La Roche Inc. No license or
immunity under any other patent is either expressed or implied by the sale of any Seegene
product.
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Table of Contents
1. Introduction -----------------------------------------------------------------------------2. Key Features ---------------------------------------------------------------------------3. List of Components -------------------------------------------------------------------4. Reagent and Equipment Be Supplied by User --------------------------------5. Storage Condition ---------------------------------------------------------------------6. Protocol for Forever 100bp Ladder Personalizer -----------------------------Appendix A. Information of Each Size Marker ------------------------------------Appendix B. Expected Result of Personalized DNA Ladder ------------------Related Products -------------------------------------------------------------------------Seegene’s Distributors -------------------------------------------------------------------
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1. Introduction
Our unique 100 bp endless usage ladder system (patent pending) is clearly
distinguished from any existing commercialized consumables 100 bp DNA ladder. This
system supplies templates (plasmids) which will be used to amplify size markers. Since
12 differently sized fragments were cloned into plasmids, it has a unique feature of
endless usage with this system. Another feature is that the customer is able to select
the specific size of their interests for generating their own personalized size markers.
Also, since this system consists of 12 different genes for 12 differently sized fragments,
these genes can be used as control probes for your other research.
2. Key Features
♦ Lifetime usage: sufficient template and primers are supplied to provide enough
markers for about a 20 year supply in the typical laboratory. If needed, the plasmids
can be transformed to yield an unlimited supply of templates and the primer sequences
are supplied so that you can replace them if necessary.
♦ Ready-PCR Format: 12 different plasmids are supplied in ready-to-PCR use
conditions. The concentration of each template (plasmid) and PCR conditions are set
to amplify all 12 fragments in the same reaction to generate a ladder of approximately
equal intensities, with a couple at higher concentration for orientation. This can be
done to generate a quick ladder, albeit without accurately knowing amount of each
band as is obtained when the mix is generated after quantitation of each PCR product.
Simply amplify each of 12 differently sized fragments by using the Ready-PCR format
plasmids and mix them together. Any further steps of transformation, plasmid preps,
restriction analysis, etc. are not needed.
♦ Able to create your own personalized ladder: to create your own personalized
ladder, simply use the two included universal primers and 12 individual templates to
amplify each of 12 differently sized fragments, quantitate, and mix according to your
requirements.
♦ Able to use differently size fragments as control probes: 12 differently sized
fragments consist of different kinds of genes such as p53, GAPDH, C-myc, C-fos, TNF,
NOS, CD4, IFN, GM-CSF, IL-1α, SCI/MIP-1α, and Beta 2-microglobulin. These genes
can be used as control probes in your other research such as hybridization-related
experiments. In addition, this ladder can be radiolabeled using T4 DNA polymerase.
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♦ Save $$$$$$: see the table below and figure out how much money you will save!
Products
Forever 100bp
Ladder Personalizer
(Seegene, Inc)
All existing 100bp
DNA Ladder
Features
(Other Companies)
No
Lifetime usage
Yes
Yes
Consumable
No
No
Personalizable
Yes
$1,788 ~ $2,336 per year
Cost
$175 for lifetime usage
* This amount is calculated in the event that 250 µg of 100 bp ladder is consumed per
every 3 months
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3. List of Components
12 DNA ladder plasmids (each 125 rxns)
10 µM primer 1 (300 rxns): 5’-GTCTACCAGGCATTCGCTTCAT-3’
10 µM primer 2 (300 rxns): 5’-CTGTGAATGCTGCGACTACGAT-3’
6 X Loading dye (1 ml)
4. Reagent and Equipment Be Supplied by User
2 mM dNTP
Thermal cycler
Taq polymerase
5. Storage Conditions
Store reagents below –20C. Plasmid DNA is stable at least one year at –20C.
Avoid multiple thaw/freeze.
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6. Protocol for Forever 100bp Ladder Personalizer
The Forever 100bp Ladder Personalizer consists of 12 different templates (plasmids)
as Ready-PCR Format. PCR reaction can be performed individually using each
template according to your requirements, and then mix the PCR products together.
1. Add the following reagents to a tube for PCR.
2 µl
Template
5 µl
10X PCR buffer (MgCl2 free)
5 µl
25 mM MgCl2
? µl
10 µM Primer 1*
? µl
10 µM Primer 2*
5 µl
2 mM dNTP
0.5 µl
Taq DNA polymerase (5 U/µl)
? µl
DW
50 µl
Total
* According to each size marker, the concentration of primer differs.
Size
Primer 1 Primer 2
marker
100~300bp
3 µl
3 µl
400~500bp
2 µl
2 µl
600~1500bp
1 µl
1 µl
2. Commence the PCR reaction using following program.
No. of
Segment cycles Temperature
Duration
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94 ℃
5 min
1
94 ℃
40 sec
35
68 ℃
40 sec
2
72 ℃
40 sec
1
72 ℃
5 min
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3. Electrophorese 0.5~ 1 µl of PCR products on 2% agarose gel stained with EtBr.
4. Mix PCR products together.
5. Use ? dosage of the mixture as personalized DNA size marker.
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Appendix A. Information of Each Size Marker
The 12 different plasmids are supplied in ready-to-PCR use conditions. Simply amplify
the 12 differently sized fragments using the plasmids and mix them together. The 12
different plasmids consist of different kinds of genes isolated from mouse (ICR) tissues.
These genes can be used as control probes in your other research such as
hybridization-related experiments.
size
100bp
200bp
300bp
400bp
500bp
600bp
700bp
800bp
900bp
1000bp
1200bp
1500bp
Gene Information
Inducible NOS
CD4
P53 tumor suppressor
TNF-α
GAPDH
c-myc
Interferon-α
β2-Microglobulin
SCI/MIP-1a
Interleukin-1α
GM-CSF
c-fos
Fig. 1. Photograph of 100 bp DNA ladder fragments
synthesized by PCR reaction using each ladder plasmid as a template. Lane 1:
100bp DNA ladder with high intensity at 500 and 1000bp. Lane 2: 100bp DNA ladder
with same intensity. Lanes 3 to 14 are shown each PCR product as a differently sized
marker.
Appendix B. Expected Result of Personalized DNA Ladder
To create your own personalized ladder, simply choose your desired plasmid templates
each having differently sized insert, amplify them using the two included universal
primers, and mix them.
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Fig. 2. Examples of 100 bp personalized DNA ladder.
Agarose gel electrophoresis is shown the 100 bp DNA ladder
generated by using Forever 100 bp Ladder Personalizer.
Lane 1 : 100 bp DNA ladder (100 ~ 1500 bp). Lane 2 : 200,
400, 600, 800, 1000 and 1500 bp. Lane 3 : 100, 300, 500,
700, 900, and 1200 bp. Lane 4 : 100, 200, 300, 400, and 500
bp. Lane 5 : 500, 600, 700, 800, 900, 1000, 1200, and 1500
bp.
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