SequaMark DNA Size Marker For sequencing and microsatellite marker sizing Catalog nos. 701001, 701004, 701005, 701006 Version B July 25, 2002 25-0565 www.invitrogen.com tech_service@invitrogen.com
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Contents and Storage Types of Products This manual is supplied with the following products: Product Catalog no. SequaMark for isotopic methods 701001 SequaMark for FAM-based methods 701004 SequaMark for TAMRA-based methods 701005 SequaMark for Cy-5-based methods 701006 Components The SequaMark DNA Size Marker includes primer, template, and manual. Isotopic and non-isotopic sequencing reaction kits are available from other companies. Using standard sequencing protocols, sufficient SequaMark DNA Size Marker template and primer supplied for 400 lanes. SequaMark template DNA SequaMark template DNA is supplied lyophilized and is dissolved in high performance liquid chromatography (HPLC)-grade water before use. Base the amount of water on the sequencing kit protocol. SequaMark sequencing primer SequaMark sequencing primer is supplied lyophilized and is dissolved in HPLC-grade water before use. Base the amount of water on the sequencing kit protocol. Storage The SequaMark DNA Size Marker is shipped at ambient temperature. Store the marker -20 C after reconstitution. 3
Overview Description SequaMark is a DNA size marker designed for sequencing and microsatellite analysis. Generated with the template and primer, the marker is used in standard sequencing reactions with dideoxythymidine triphosphate (ddt) as the exclusive terminator. SequaMark markers are available for isotopic or non-isotopic sequencing methods. The reaction produces a ladder with bands appearing every 10 bases, beginning twenty bases from the special sequencing primer supplied and extending to 500 bases along the ladder. The following additional bands are present (refer to the image on page 8). Five sequential bands 100 bases past the primer (the fifth is base 100) Two sequential bands at 200 bases from the primer Three sequential bands at 300 bases from the primer Four sequential bands at 400 bases from the primer Six bands every other base at 500 bases from the primer (the first of these is base 490, and the sixth is base 500) Functions The SequaMark DNA Size Marker has the following functions: Helps to orient bands appearing in neighboring lanes on gels. Serves as a reference point for proper scoring of sequences and allows sizing of microsatellite markers. Serves as a general control for the quality of the sequencing reactions and resolving power of each gel run. Sequencing System Compatibility SequaMark works with the following sequencing systems. Sequitherm EXCEL II DNA Sequencing Kit from Epicentre using cycle sequencing with both end-labeled and internal-labeled primers fmol DNA Cycle Sequencing System from Promega Corporation Sequenase Version 2.0 Sequencing System from United States Biochemical (USB) using extension primer labeling However, SequaMark works with most sequencing kits employing a conventional Sanger sequencing method. 4
Using SequaMark DNA Size Marker To obtain the SequaMark ladder, configure the annealing reaction with the SequaMark template and SequaMark primer according to the sequencing kit instructions. The SequaMark template is single-stranded, so employ the appropriate protocol. Running the dda, ddg, and ddc reactions is unnecessary. When the sequencing reaction is complete, add stop solution according to the manufacturer s instructions. Generating a Ladder to Estimate the Size of FAM-, TAMRA-, and Cy-5-Labeled DNA Fragments The protocol described below is for the SequiTherm EXCEL II DNA Sequencing Kit (from Epicentre). Creating the Premix 1 Thaw the reagents on ice, and vortex the reagent tubes. 2 With 48 µl deionized water, dilute the template to 50 fmoles / µl. With 200 µl deionized water, dilute the primer to 1 pmole / µl. 3 Combine the following components in a 0.5 ml microcentrifuge tube labeled premix. 1.5 pmoles of dye-labeled primer 7.2 µl of SequiTherm EXCEL II Sequencing Buffer 5 to 50 fmoles of DNA template Deionized water to 16 µl 1 µl of SequiTherm EXCEL II DNA Polymerase (5 U / µl) 4 Mix the components and chill the tube on ice. 5 In the 0.5 ml tube on ice, add 2 µl of T SequiTherm EXCEL II Termination Mix. Configuring the Reaction 6. On ice, add 4 µl of the premix to the tube and mix the contents thoroughly. 7. Overlay the reaction with mineral oil and pulse centrifuge the tubes to separate the mineral oil layer from the reaction layer. Thermal Cycling 8. Preheat the thermal cycler to 95 C. 9. Heat the reaction for five minutes at 95 C. 10. Cycle the reaction 30 times for 30 seconds at 95 C 30 seconds at 52 C 60 seconds at 70 C Stopping the Reaction 11. Add 3 µl of Stop / Loading Buffer to each reaction. Proceed with electrophoresis, or store at -20 C. Continued on next page 5
Using the SequaMark DNA Size Marker, Continued Generating a Ladder to Estimate the Size of Isotope-Labeled DNA Fragments For SequiTherm EXCEL II DNA Sequencing Kit (from Epicentre) Use Cycle Sequencing Internal Labeling [α- 32 P]-dATP or Cycle Sequencing End Labeling [γ- 32 P]-ATP. Follow the manufacturer's instructions, and include the following parameters. 1. Use 50 fmoles of template. Dilute the template with 48 µl of deionized water to yield a concentration of 50 fmoles / µl. Dilute the primer with 200 µl of deionized water to yield a concentration of 1 pmole / µl. 2. Run only the ddt reaction. 3 When cycling the reaction, add an annealing step to the 30 X cycle to create the following cycle. 30 seconds at 95 C 30 seconds at 52 C 60 seconds at 70 C For Sequenase Version 2.0 Sequencing Kit (from USB) 1 Dissolve the template ssdna in 250 µl deionized water, yielding a concentration of 0.025 µg / µl. 2 Dissolve the primer in 100 µl deionized water, yielding a concentration of 2 pmoles / µl. 3 Add the following components for the annealing reaction. Template ssdna 5 µl SequaMark primer 2 µl Sequenase reaction buffer 2 µl deionized water 1 µl Total volume 10 µl 4 Anneal the primers to the DNA by heating for 2 minutes at 65 o C, and then cooling to 35 o C over 15-30 minutes. 5 Centrifuge the tube briefly, and chill it on ice. 6 For the labeling reaction, add the following components. Annealed DNA mix (from step 3) 10 µl DTT (0.1 M) 1 µl Diluted labeling mix 2 µl Diluted Sequenase enzyme 2 µl (α- 32 P) datp (approximately 3,000 Ci / mole) 1 µl Total volume 16 µl 7 Mix and incubate the reaction at room temperature for 2-3 minutes. 8 Transfer 14 µl of the reaction mix to a tube containing 10 µl of T Termination Mixture prewarmed to 37 o C. Incubate the reaction at 37 o C for 5 minutes. 9 Add 16 µl of Stop Solution. 10 Heat at 90 o C for 5 minutes and electrophorese 3 µl per well. Continued on next page 6
Using SequaMark DNA Size Marker, Continued Generating a Ladder to Estimate the Size of Isotope-Labeled DNA Fragments, continued For fmol DNA Cycle Sequencing Kit (from Promega) Follow the manufacturer's instructions, and include the following parameters. 1 For end-labeling, use 62.5 ng template. For direct incorporation, use 125 ng template. Dilute the template with 100 µl deionized water to yield a concentration of 62.5 ng / µl. Dilute the primer with 100 µl deionized water to yield a concentration of 2 pmole / µl. 2 Run only the ddt reaction. 3 When cycling the reaction, add an annealing step to the 30 X cycle to create the following cycle. 30 seconds at 95 C 30 seconds at 58 C 60 seconds at 70 C Electrophoresing the SequaMark Marker 1. Briefly centrifuge the tubes and heat the tubes for 5 minutes at 70 C. 2. Electrophorese 1 to 4 µl of the reaction per well using the following conditions to produce the SequaMark ladder: Manual Sequencing 6 % acrylamide gel Electrophoresis power: 40 W Electrophoresis voltage: 1,700 ABI 377 Sequencer 4.5 % acrylamide gel Voltage: 2,400 V Current: 50 ma Power: 200 W Temperature: 51 C Laser power: 40 mw CCD gain: 2 Storing the Annealing Reaction Store the product of the annealing reaction at -20 C for up to two weeks without loss of quality. Continued on next page 7
Using SequaMark DNA Size Marker, Continued Example An example of SequaMark DNA Size marker analyzed on a 6% acrylamide-urea sequencing gel using the isotope label is shown in the image to the right. The reaction produces a ladder with bands appearing every 10 bases, beginning twenty bases from the special sequencing primer supplied and extending to 500 bases along the ladder. The following additional bands are present: Five sequential bands 100 bases past the primer (the fifth is base 100) Two sequential bands at 200 bases from the primer Three sequential bands at 300 bases from the primer Four sequential bands at 400 bases from the primer Six bands every other base at 500 bases from the primer (the first of these is base 490, and the sixth is base 500) 8
Technical Service Contact Us For more information or technical assistance, call, write, fax, or email. Additional international offices are listed on our Web page (www.invitrogen.com). Corporate Headquarters: Invitrogen Corporation 1600 Faraday Avenue Carlsbad, CA 92008 USA Tel: 1 760 603 7200 Tel (Toll Free): 1 800 955 6288 Fax: 1 760 602 6500 E-mail: tech_service@invitrogen.com Japanese Headquarters: Invitrogen Japan K.K. Nihonbashi Hama-Cho Park Bldg. 4F 2-35-4, Hama-Cho, Nihonbashi Tel: 81 3 3663 7972 Fax: 81 3 3663 8242 E-mail: jpinfo@invitrogen.com European Headquarters: Invitrogen Ltd Inchinnan Business Park 3 Fountain Drive Paisley PA4 9RF, UK Tel: +44 (0) 141 814 6100 Tel (Toll Free): 0800 5345 5345 Fax: +44 (0) 141 814 6117 E-mail: eurotech@invitrogen.com 2001-2002 Invitrogen Corporation. All rights reserved. Sequenase is a trademark of United States Biochemical. Sequitherm EXCEL II is a trademark of Epicentre. fmol is a registered trademark of Promega Corporation. 9