User Manual. For Research Use Only. Catalog No. FMLP Storage Conditions: -20 o C. Version 1.0 Published January 2004

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1 Forever Multi-Ladder Personalizer I User Manual Version 1.0 Published January 2004 Catalog No. FMLP-2004 Storage Conditions: -20 o C For Research Use Only

2 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 BioGene Ltd 6 The Business Centre, Harvard Way Kimbolton, Cambridgeshire PE28 0NJ Tel: +44(0) Fax: +44(0) info@biogene.com URL: 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. 2

3 Table of Contents 1. Introduction Key Features List of Components Reagent and Equipment to be Supplied by User Storage Condition Protocol for Forever Multi-Ladder Personalizer I Appendix A. Information of Each Size Marker Appendix B. Expected Result of Personalized DNA Ladder Related Products

4 1. Introduction Our unique endless usage Multi-ladder system (patent pending) is clearly distinguished from any existing commercialized consumables 50bp and 100bp DNA ladders. This system supplies templates (plasmids) which will be used to amplify size markers. Twelve different useful genes were used as templates for each fragment of the 50bp/ 100bp DNA ladders ranging in size from 100bp to 1.55kb, respectively. 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, 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 years in a 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 24 fragments in the same reaction condition 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 24 differently sized fragments by using the Ready-PCR format plasmids and mix them together. Able to create your own personalized ladder: to create your own personalized ladder, simply use the each primer combinations and 12 individual templates to amplify each of 24 differently sized fragments, quantitate, and mix according to your requirements. Able to use differently size fragments as control probes: 12 different plasmids consist of different kinds of genes such as p53, GAPDH, C-myc, C-fos, TNF, NOS, CD4, IFN, GM-CSF, IL-1α, SCI/MIP-1a, 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. 4

5 Save s: see the table below and figure out how much money you will save! Features Products Forever Multi-Ladder Personalizer I (Seegene, Inc) All existing 100bp DNA Ladder (Other Companies) Lifetime usage Yes No Consumable No Yes Personalizable Yes No Cost Single time purchase for lifetime usage 981 ~ 1,281 per year * This amount is calculated in the event that 250 µg of 100bp ladder is consumed per every 3 months Forever DNA Size Marker Series Product Cat. # Ladder type Size Forever 100bp Ladder Personalizer FBLP bp ladder 12 fragments Forever Multi-Ladder Personalizer I FMLP /50bp ladders 24 fragments 5

6 3. List of Components 12 DNA ladder plasmids (each 50 rxns) 10 µm primer 1 for 100bp ladder (300 rxns): 10 µm primer 2 for 100bp ladder (300 rxns): 10 µm primer 3 for 50bp ladder (150 rxns): 10 µm primer 4 for 50bp ladder (150 rxns): 6 X Loading dye (1 ml) 4. Reagent and Equipment to be Supplied by User 2 mm dntp Thermal cycler Taq polymerase 5. Storage Conditions Store reagents below 20. Plasmid DNA is stable at least one year at 20. Avoid multiple thaw/freeze. 6. Protocol for Forever Multi-Ladder Personalizer I The Forever Multi-Ladder Personalizer I 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 (MgCl 2 free) 5 µl 25 mm MgCl µl 10 µm Primer 1 or 3 * 1-3 µl 10 µm Primer 2 or 4 * 5 µl 2 mm dntp 0.5 µl Taq DNA polymerase (5 U/µl) µl Distilled water 50 µl Total volume 6

7 * According to each size marker, the concentration of primer differs. Template (plasmid) Primer 1 Primer 2 Primer combination 1 Primer combination 2 Expected Primer 3 Primer 4 size Expected size 1 3 µl 3 µl 100bp 3 µl 3 µl 150bp 2 3 µl 3 µl 200bp 3 µl 3 µl 250bp 3 3 µl 3 µl 300bp 3 µl 3 µl 350bp 4 2 µl 2 µl 400bp 2 µl 2 µl 450bp 5 2 µl 2 µl 500bp 2 µl 2 µl 550bp 6 1µl 1 µl 600bp 1µl 1 µl 650bp 7 1µl 1 µl 700bp 1µl 1 µl 750bp 8 1µl 1 µl 800bp 1µl 1 µl 850bp 9 1µl 1 µl 900bp 1µl 1 µl 950bp 10 1µl 1 µl 1000bp 1µl 1 µl 1050bp 11 1µl 1 µl 1200bp 1µl 1 µl 1250bp 12 1µl 1 µl 1500bp 1µl 1 µl 1550bp Note: The final concentration of MgCl 2 could be adjustable depending on the commercial brand of Taq DNA polymerase. Please refer to each instruction of Taq DNA polymerase. Note: You can use a general Taq DNA polymerase. Among the commercialized DNA polymerase which we have had especially experience, we have received good results with Promega s (Cat. No. M1661), Roche s (Cat. No ), Invitrogen s (Cat. No ), and Takara s Taq DNA polymerase (Cat. No. R001AM). 2. Commence the PCR reaction using following program. Segment No. of cycles Temperature Duration min sec sec sec min 3. Run µl of each PCR product on 2% agarose gel stained with EtBr. 4. Mix PCR products together after they were synthesized successfully. 5. Use proper dosage of the mixture to personalize a DNA size marker. Note: Usually you can obtain loading reactions per one PCR reaction. 7

These genes can be used as control probes in your other research such as hybridization-related experiments.

8 Appendix A. Information of Each Size Marker The 12 different plasmids are supplied in ready-to-pcr use conditions. Simply amplify the 24 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 Gene Information 100, 150bp Inducible NOS 200, 250bp CD4 300, 350bp P53 tumor suppressor 400, 450bp TNF-α 500, 550bp GAPDH 600, 650bp c-myc 700, 750bp Interferon-α 800, 850bp β2-microglobulin 900, 950bp SCI/MIP-1a 1000, 1050bp Interleukin-1α 1200, 1250bp GM-CSF 1500, 1550bp 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. Fig. 2. Various types of customized DNA ladders using Forever Multi-Ladder Personalizer I. Lane 1: 100bp to 1000bp in 50nt increments, 1200, 1250, 1500, and 1550bp Lane 2: 100bp to 1000bp in 100nt increments, 1200, and 1500bp Lane 3: 100, 150, 200, 250, 300, 350, 450, 500, 550, 600, 650, 700, 750, 800, and 1500bp Lane 4: 100, 200, 300, 400, and 500bp Lane 5: 100, 150, 200, 250, 300, 350, 450, and 500bp Lane 6: 500, 600, 700, 800, 900, 1000, 1200, and 1500bp Lane 7: 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1200, and 1500bp 8

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9 Related Products GeneFishing TM DEG kits All of the GeneFishing DEG kits (DEG101~106) comprise 20 ransdomly selected arbitrary ACPs (Annealing Control Primers) and each DEG kit works equally for your target samples. Full-length cdnas Seegene's Full-length cdnas are ideal to study gene expression in specific tissues and at specific developmental stages and also to clone the genes belonging to a multigene family. Pre-made Northern Blots Northern blots are pre-made for immediate use and designed to See Gene expression in specific tissues and at specific developmental stages. Our Northern blots allow you to assess the distribution, size, alternative splicing forms, and level of your transcripts in one experiment. Zoo Blot We are offering zoo blot(pre-made Southern blot) including 12 different species for your screening assays. Genomic DNAs were prepared from human, rat(sd), mouse(icr), dog, cow, pig, rabbit, chicken, frog(xenops), fish(zebra fish), C. elegans, and yeast. Genomic DNAs We are offering genomic DNAs obtained from 12 different sample sources for your screening assays. Seegene's genomic DNA is qualified for genomic analysis including PCR and library construction DNA Walking SpeedUp TM Kit As a third commercial application of Seegene's proprietary ACP (Annealing Control Primer) Technology maximizing PCR specificity, DNA Walking SpeedUp Kit is directed to the method using our unique DNA Walking ACP (DW-ACP) primer designed to capture unknown target sites and the optimized PCR conditions. 9

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