Manuale d’uso / di manutenzione del prodotto 500 reactions del fabbricante GE
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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.
2 CONTENTS Components of the Kit ....................................................................................... 3 Quality Control .................................................................................................... 4 Safety Warnings and Precautions .
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.
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.
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).
6 Chain termination sequencing This kit is designed to eliminate sequencing artifacts such as stops (or BAFLs— bands across four lanes) and background bands.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 Commun.
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.
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 .
Un punto importante, dopo l’acquisto del dispositivo (o anche prima di acquisto) è quello di leggere il manuale. Dobbiamo farlo per diversi motivi semplici:
Se non hai ancora comprato il GE 500 reactions è un buon momento per familiarizzare con i dati di base del prodotto. Prime consultare le pagine iniziali del manuale d’uso, che si trova al di sopra. Dovresti trovare lì i dati tecnici più importanti del GE 500 reactions - in questo modo è possibile verificare se l’apparecchio soddisfa le tue esigenze. Esplorando le pagine segenti del manuali d’uso GE 500 reactions imparerai tutte le caratteristiche del prodotto e le informazioni sul suo funzionamento. Le informazioni sul GE 500 reactions ti aiuteranno sicuramente a prendere una decisione relativa all’acquisto.
In una situazione in cui hai già il GE 500 reactions, ma non hai ancora letto il manuale d’uso, dovresti farlo per le ragioni sopra descritte. Saprai quindi se hai correttamente usato le funzioni disponibili, e se hai commesso errori che possono ridurre la durata di vita del GE 500 reactions.
Tuttavia, uno dei ruoli più importanti per l’utente svolti dal manuale d’uso è quello di aiutare a risolvere i problemi con il GE 500 reactions. Quasi sempre, ci troverai Troubleshooting, cioè i guasti più frequenti e malfunzionamenti del dispositivo GE 500 reactions insieme con le istruzioni su come risolverli. Anche se non si riesci a risolvere il problema, il manuale d’uso ti mostrerà il percorso di ulteriori procedimenti – il contatto con il centro servizio clienti o il servizio più vicino.
