GE AH9539 Uživatelský manuál Strana 1

Thermo SequenaseRadiolabeled TerminatorCycle Sequencing KitProduct Number 79750, 50 reactions79760, 100 reactions79770, 500 reactionsProduct Number 18

103. Cycling termination reactionsTransfer 4.5µl of reaction mixture (prepared in step 2) to each terminationtube (‘G’, ‘A’, ‘T’ and ‘C’) from step 1.

11SUPPLEMENTARY INFORMATIONGeneral guidelines• Since the popular multiple cloning sites all derive from similar sequences,one primer can serve for the

12Preparation of template DNASince cycle sequencing can be performed using very little template DNA, onlyvery small amounts of detrimental impurities

13As another example, when using the universal -40 17-mer, which has a meltingtemperature of about 50°C, cycling between 45°C and 95°C is effective. I

140.5pmol 1pmol 2pmol 8pmol300 bases150 basesFigure 1. Excess template DNA can reduce sequence extension lengths. In cases where2pmol or more template

15Sequencing PCR ProductsThe products of Polymerase Chain Reaction (PCR) can have structures whichmake them difficult to sequence. One of the most com

16A suitable nucleotide mixture containing dITP is included in the kit for use withtemplates prone to gel compression artifacts. To use dITP simply su

17Figure 3. Use of dITP requires longer extension times at 60°C. Shown are foursequences of plasmid pUC18 obtained using cycles with 1, 4, 10 and 20 m

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

19dilute—approximately 0.8X strength. Be certain to run glycerol tolerant gels atthe same power (wattage) as TBE-buffered gels so the gel temperature

2CONTENTSComponents of the Kit ...3Quality Control ...

20General guidelines for electrophoresis1. Ultrapure or electrophoresis grade reagents should be used.2. Sequencing gels should be made fresh. Store s

21than 1pmol of template DNA for each sequence (0.25pmol per reaction).See figure 1.3. G-C rich template producing strong secondary structure. Try les

22spaced, a compression artifact is indicated. Try using the dITP reactionmixture or a formamide gel.Bands in 2 or 3 lanes1. Heterogeneous template DN

23REFERENCES1. 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

24RELATED PRODUCTSKits and EnzymesProduct Application Pack size Product numberSequenase PCR Product For rapid sequencing 100 70170Sequencing Kit of PC

25Asia PacificTel: +852 2811 8693AustraliaTel: +61 2 9894 5188AustriaTel: +43 1 57 606 1610BelgiumTel: 0800 73888CanadaTel: 1 800 463 5800Central and

26Material safety data sheetRevision: 10/30/00Hazard information is provided for compliance with both the UKChemicals (Hazard Information and Packagin

27HAZARDS IDENTIFICATION CHIPFormamide: Toxic to reproduction, category 3. Tris: Irritant.HCSFormamide: Teratogen. Tris-HCl, Tris and EDTA: Irritant.F

All goods and services are sold subject to the terms and conditions of sale of thecompany within the USB Corporation or the group which supplies them.

3COMPONENTS OF THE KITThe solutions included in the Thermo Sequenase™ Radiolabeled TerminatorCycle Sequencing Kit have been carefully prepared to yiel

4Redivue nucleotides can be stored at 4°C for up to 1 week after receipt, orat a constant -20°C if longer storage is desired. Care must be taken topre

5INTRODUCTIONThis sequencing kit combines two revolutionary innovations for sequencingDNA using radioactive labels. First, the label is incorporated i

6Chain termination sequencingThis kit is designed to eliminate sequencing artifacts such as stops (or BAFLs—bands across four lanes) and background ba

7uses dideoxynucleoside triphosphates, generating uniform band intensities insequencing experiments (with dGTP). These properties make the enzyme idea

8MATERIALS NOT SUPPLIEDNecessary reagents:Water—Only deionized, distilled water should be used for the sequencingreactions.Specialized sequencing prim

9PROTOCOL1. Termination mixes—Prepare the termination mixes on ice. Mix 2µl ofNucleotide Master Mix (either dGTP or dITP—see note below) and 0.5µl of[