GE 188403 manuel d'utilisation

Thermo Sequenase Radiolabeled T erminator Cyc le Sequencing Kit Pr oduct Number 79750, 50 reactions 79760, 100 reactions 79770, 500 reactions Pr oduct Number 188403 inc ludes: 79750, 50 reactions AH9539, 33 P-labeled terminator s ST ORA GE Store at -15 ° C to -30 ° C. Warning: For research use only. Not recommended or intended for diagnosis of di[...]

2 CONTENTS Components of the Kit ....................................................................................... 3 Quality Control .................................................................................................... 4 Safety Warnings and Precautions .............................................................. 4, 26 Introdu[...]

3 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 [...]

4 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 observ[...]

5 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 th[...]

6 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 termi[...]

7 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 [...]

8 MA TERIALS NO T 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 sequ[...]

9 PRO T OCOL 1. Termination mixes —Prepare the termination mixes on ice. Mix 2 µ l of Nucleotide Master Mix (either dGTP or dITP—see note below) and 0.5 µ l of [ α - 33 P]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 2.5 ?[...]

10 3. Cycling termination reactions Transfer 4.5 µ l of reaction mixture (prepared in step 2) to each termination tube (‘G’, ‘A’, ‘T’ and ‘C’) from step 1. Mix well and overlay with 10-20 µ l of mineral oil (if needed). Cap and place the tube in the thermal cycling instrument. Note: When sequencing single-stranded DNA, the primer [...]

11 SUPPLEMENT AR Y INFORMA TION 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. Among the vectors compatible with the primer supplied in the Thermo Sequenase radiolabeled terminator cycle sequencing kit are M13mp8,[...]

12 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-s[...]

13 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 95-9[...]

14 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 16 µ g (8pmol) of M13mp18 DNA[...]

15 Sequencing PCR Pr oducts 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 PC[...]

16 A suitable nucleotide mixture containing dITP is included in the kit for use with templates prone to gel compression artifacts. To use dITP simply substitute the dITP Nucleotide Mix for the dGTP Nucleotide Mix. All other aspects of the sequencing protocol remain unchanged except that when using dITP, reduce the termination temperature from 72 °[...]

17 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. Reading farther from or c loser to the primer The termination mixes descr[...]

18 by 50%, thus increasing the average extension length of each primer before a ddNTP is incorporated. Conversely, adding 1 µ l of [ α - 33 P]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 [...]

19 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 Na 2 EDTA . 2H 2 O 9.3g H 2 O to 1000ml, filter (may be autoclaved) This is the traditional sequencing gel buffer. It should NOT be [...]

20 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 acc[...]

21 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 fac[...]

22 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 sequ[...]

23 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 [...]

24 RELA TED PRODUCTS Kits and Enzymes Product Application Pack size Product number Sequenase PCR Product For rapid sequencing 100 70170 Sequencing Kit of PCR products templates Sequenase Quick-Denature For rapid denaturation 100 70140 Plasmid Sequencing Kit and sequencing of templates plasmid DNA Sequenase Version 2.0 For non-cycle 200 units 70775Y[...]

25 Asia Pacific Tel: +852 2811 8693 Australia Tel: +61 2 9894 5188 Austria Tel: +43 1 57 606 1610 Belgium Tel: 0800 73888 Canada Tel: 1 800 463 5800 Central and East Europe Tel: +43 1 982 3826 Denmark Tel: +45 4516 2400 Finland Tel: +358 9 512 3940 Former Soviet Union Tel: +7 (095) 232 0250 France Tel: +33 1 69 35 67 00 Germany Tel: +49 761 49 03 0[...]

26 Material safety data sheet Revision: 10/30/00 Hazard information is provided for compliance with both the UK Chemicals (Hazard Information and Packaging) (CHIP) Regulations and the US Hazard Communication Standard (HCS) IDENTIFICATION OF THE PRODUCT NAME PRODUCT CODE EEC NUMBER SUBSTANCE/PREPARATION Thermo Sequenase Radiolabeled 79750, 79760 Non[...]

27 HAZARDS IDENTIFICATION CHIP Formamide: Toxic to reproduction, category 3. Tris: Irritant. HCS Formamide: Teratogen. Tris-HCl, Tris and EDTA: Irritant. FIRST-AID MEASURES Remove from exposure. Flush from skin or eyes with water. If irritation is evident or if ingested or inhaled, seek medical advice. FIRE-FIGHTING INFORMATION For small fires only[...]

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 . The purchase of this kit (reagent)[...]