Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit Product Number 79750, 50 reactions 79760, 100 reactions 79770, 500 reactions Product Number 188403 includes: 79750, 50 reactions AH9539, 33P-labeled terminators STORAGE Store at -15°C to -30°C. Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals.
CONTENTS Components of the Kit ....................................................................................... 3 Quality Control .................................................................................................... 4 Safety Warnings and Precautions .............................................................. 4, 26 Introduction ......................................................................................................... 5 Materials Not Supplied ...........................
COMPONENTS OF THE KIT The solutions included in the Thermo Sequenase™ Radiolabeled Terminator Cycle Sequencing Kit have been carefully prepared to yield the best possible sequencing results. Each reagent has been tested extensively and its concentration adjusted to meet USB™ standards. It is strongly recommended that the reagents supplied in the kit be used as directed. The following solutions are included in the kit: Thermo Sequenase DNA Polymerase: 4U/µl, 0.
Redivue nucleotides can be stored at 4°C for up to 1 week after receipt, or at a constant -20°C if longer storage is desired. Care must be taken to prevent evaporation of these small volumes of material. Tightly cap the vials after use. Store at -20°C between uses if frequency of use is less than every 1-3 days. If condensation is observed on the walls of the vial or in the cap, return the liquid to the bottom of the vial and mix well before use.
INTRODUCTION This sequencing kit combines two revolutionary innovations for sequencing DNA using radioactive labels. First, the label is incorporated into the DNA sequencing reaction products by the use of four [α-33 P]dideoxynucleotide (ddNTP) terminators (G,A,T,C). The labeled ddNTPs are more efficient for labeling sequencing experiments than other labeled nucleotides because they specifically label only the properly terminated DNA chains.
Chain termination sequencing This kit is designed to eliminate sequencing artifacts such as stops (or BAFLs— bands across four lanes) and background bands. BAFLs can result from the enzyme pausing at regions of secondary structures in GC-rich templates, producing prematurely aborted primer extension products of the same length in all four termination reactions.
uses dideoxynucleoside triphosphates, generating uniform band intensities in sequencing experiments (with dGTP). These properties make the enzyme ideal for generating high-quality DNA sequences using cycle-sequencing methods. It is stable at 90°C for at least 1 hour and retains 50% of its activity when incubated at 95°C for 60 minutes. The Thermo Sequenase polymerase in this kit combines the advantages of both Sequenase DNA polymerase and Taq DNA polymerase.
MATERIALS NOT SUPPLIED Necessary reagents: Water—Only deionized, distilled water should be used for the sequencing reactions. Specialized sequencing primers—Some sequencing projects will require the use of primers which are specific to the project. For most sequencing applications, 0.5-2.5pmol of primer should be used for each set of sequencing reactions. Always determine the concentration of the primer by reading the optical density at 260nm (OD260).
PROTOCOL 1. Termination mixes—Prepare the termination mixes on ice. Mix 2µl of Nucleotide Master Mix (either dGTP or dITP—see note below) and 0.5µl of [α-33P]ddNTP (G, A, T, or C—one of each per sequence) to produce a termination mix for each ddNTP. Label, fill and cap four tubes (‘G’, ‘A’, ‘T’, ‘C’) with 2.5µl of each termination mix. It is more accurate and convenient to prepare batches of termination mixes sufficient for all sequences to be performed, then dispense 2.
3. Cycling termination reactions Transfer 4.5µl of reaction mixture (prepared in step 2) to each termination tube (‘G’, ‘A’, ‘T’ and ‘C’) from step 1. Mix well and overlay with 10-20µl of mineral oil (if needed). Cap and place the tube in the thermal cycling instrument. Note: When sequencing single-stranded DNA, the primer may anneal to the template with reduced specificity while the tubes are on ice, and extension of these primers can occur as the thermal cycler heats up during the first cycle.
SUPPLEMENTARY INFORMATION General guidelines • Since the popular multiple cloning sites all derive from similar sequences, one primer can serve for the sequencing of insert DNA in most of the common vectors.
Preparation of template DNA Since cycle sequencing can be performed using very little template DNA, only very small amounts of detrimental impurities are likely to be carried along with the DNA. Therefore, though still important, template purity may not be as crucial for cycle sequencing as it is for non-cycle sequencing. Preparation of single-stranded template DNA Single-stranded template DNA of good purity is essential for excellent sequencing results.
As another example, when using the universal -40 17-mer, which has a melting temperature of about 50°C, cycling between 45°C and 95°C is effective. If in doubt, choose a wide temperature range with pauses (15-30 seconds) at the extremes of temperature. The termination reaction cycles should always have a denaturation temperature of 95-98°C (however, avoid extended steps at 98°C since at this temperature the enzyme has a half-life of less than one hour).
0.5pmol 1pmol 2pmol 8pmol 300 bases 150 bases Figure 1. Excess template DNA can reduce sequence extension lengths. In cases where 2pmol or more template DNA are sequenced, the supply of nucleotides can be exhausted before extensions reach suitable length for optimal sequencing. These sequences were run using up to 16µg (8pmol) of M13mp18 DNA template.
Sequencing PCR Products The products of Polymerase Chain Reaction (PCR) can have structures which make them difficult to sequence. One of the most common problems associated with sequencing of PCR products is the presence of stops or BAFLs, where the sequence pauses or stops at artifactual ends in the template (actually the ends of truncated PCR product).
dGTP → dITP dGTP dITP → Figure 2. Compression artifacts can be eliminated using dITP in place of dGTP without interference by stops or other artifact bands. Shown are two severely ‘compressed’ regions of secondary structure (see arrows). The sequences run using dITP in place of dGTP are accurate and unambiguous. A suitable nucleotide mixture containing dITP is included in the kit for use with templates prone to gel compression artifacts.
1min 4min 10min 20min 300 bases 200 bases 150 bases Figure 3. Use of dITP requires longer extension times at 60°C. Shown are four sequences of plasmid pUC18 obtained using cycles with 1, 4, 10 and 20 minute extension steps in the cycles. Extension steps of 4-5 minutes or longer are necessary for reading beyond 200 bases.
by 50%, thus increasing the average extension length of each primer before a ddNTP is incorporated. Conversely, adding 1µl of [α-33P]ddNTP will decrease the ratio by 50%, thus decreasing the average extension length of each primer. Running sequencing gels which resolve more than 600 nucleotides requires high quality apparatus, chemicals and attention to many details.
dilute—approximately 0.8X strength. Be certain to run glycerol tolerant gels at the same power (wattage) as TBE-buffered gels so the gel temperature is normal. 10X TBE Buffer (70454) Tris base 108g Boric acid 55g 9.3g Na2EDTA.2H2O H2O to 1000ml, filter (may be autoclaved) This is the traditional sequencing gel buffer. It should NOT be used with the polymerase supplied in this kit (Glycerol Tolerant Gel Buffer should be used). Gel recipes (for 100ml of gel solution) Standard gel Gel conc.
General guidelines for electrophoresis 1. Ultrapure or electrophoresis grade reagents should be used. 2. Sequencing gels should be made fresh. Store solutions no longer than one week in the dark at 4°C. Commercial preparations of acrylamide gel mixes in liquid or powder form (RapidGel gel mixes—see ‘Related Products’) should be used according to manufacturers recommendations. 3. Gels should be prepared 2-20 hours prior to use, and pre-run for ~15 minutes. 4.
than 1pmol of template DNA for each sequence (0.25pmol per reaction). See figure 1. 3. G-C rich template producing strong secondary structure. Try less DNA, longer extension times, more cycles, more enzyme, 5% DMSO, or a 96°C denaturation temperature. Film blank or very faint 1. If using single-sided film, the emulsion side must be placed facing the dried gel. 2. DNA preparation may be bad. Try the control DNA supplied in the kit. 3. Labeled dideoxynucleotide too old. Try longer exposure. 4.
spaced, a compression artifact is indicated. Try using the dITP reaction mixture or a formamide gel. Bands in 2 or 3 lanes 1. Heterogeneous template DNA (2 bands) caused by spontaneous deletions arising during M13 phage growth. Try control DNA and limit phage growth to less than 6-8 hours. 2. Insufficient mixing of reaction mixtures. 3. The sequence may be prone to compression artifacts in the gel.
REFERENCES 1. SANGER, F., NIKLEN, S., and COULSON, A.R. (1977) Proc. Nat. Acad. Sci. USA 74, pp 5463-5467. 2. BIGGIN, M.D., GIBSON, T.J., and HONG, G.F. (1983) Proc. Nat. Acad. Sci. USA 80, pp 3963-3965. 3. ZAGURSKY, R.J., CONWAY, P.S., and KASHDAN, M.A. (1991) BioTechniques 11, pp 36-38. 4. ANSORGE, W., and LABEIT, S. (1984) J. Biochem. and Biophys. Method 10, pp 237-243. 5. SHEEN, J., and SEED, B. (1988) BioTechniques 6. 6. TABOR, S., and RICHARDSON, C.C. (1987) Proc. Nat. Acad. Sci. USA 84, pp 4767-4771.
RELATED PRODUCTS Kits and Enzymes Product Sequenase PCR Product Sequencing Kit Sequenase Quick-Denature Plasmid Sequencing Kit Sequenase Version 2.0 DNA polymerase Sequenase Version 2.
Product RapidGel-GTG-8% RapidGel-40% RapidGel-XL-6% RapidGel-XL-8% RapidGel-XL-40% TBE Buffer, 10X TEMED Application Gel electrophoresis Gel electrophoresis Gel electrophoresis Gel electrophoresis Gel electrophoresis Gel electrophoresis Gel electrophoresis Tris Gel electrophoresis Urea Gel electrophoresis Water, RNase-free X-Gal Xylene cyanol Cloning Gel electrophoresis Pack size 500ml 500ml 500ml 500ml 500ml 6 bottles 100g 500g 5kg 1kg 1kg 500g 500ml 1 liter 250mg 25g Product number 75847-500ml 75
COMPOSITION/ HAZARDOUS COMPONENTS PRODUCT CODE EEC NUMBER 79750, 79760 None CAS NO. 75-12-7 1185-53-1 77-86-1 6381-92-6 HAZARD Formamide in 70724 Tris-HCl in Part 79802 Tris in Part 71949 EDTA in Part 71949 For the Part 71949 mixture/preparation 1.4% 74% 4.1% 96% %WT — — — 10ppm TLV n Formamide R:36/38 Irritating to eyes and skin S:26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
For small fires only: Use carbon dioxide, dry powder or foam. Wear suitable protective clothing including lab coat, safety glasses and gloves to clean small releases. Wear suitable protective clothing including lab coat, safety glasses and gloves. Store at -20°C. Same as handling and storage information. Pregnant women or women of child bearing age should minimize contact and exposure to formamide. Molecular biology sequencing kit containing separate vials and solutions of reagents. Product is stable.
All goods and services are sold subject to the terms and conditions of sale of the company within the USB Corporation or the group which supplies them. A copy of these terms and conditions is available on request. ‡Notice to purchaser about limited license This product is sublicensed from GE Healthcare UK Ltd.
