Benutzeranleitung / Produktwartung RPN1607 des Produzenten GE
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GE Healthcare Amersham Megaprime™ DNA Labelling Systems Product Booklet Codes: RPN1604 RPN1605 RPN1606 RPN1607.
2 Page finder 1. Legal 3 2. Handling 4 2.1. Safety warnings and precautions 4 2.2. Storage and stability 4 2.3. Quality control 4 3. System components 6 3.1. Megaprime DNA labelling systems 8 4. Introduction 9 5. Megaprime DNA labelling protocols 11 5.
1. Legal GE and GE monogram are trademarks of General Electric Company. Amersham, Megaprime, Hybond, Hyperfilm, Hypercassette, Hyperscreen, Sensitize, Sephadex and SepRate are trademarks of GE Healthcare companies. © 2006 General Electric Company – All rights reserved.
4 2. Handling 2.1. Safety warnings and precautions Warning: For r esearch use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. Caution: For use with radioactive material.
5 using 17 pmol/25 ng DNA of [ α – 32 P] labelled nucleotides, specific activity 3000 Ci/mmol (codes PB 10204-7) and RPN 1606/1607 are tested using 17 pmol/25 ng DNA of [ α – 32 P]dCTP , 3000 Ci/mmol (code PB 10205).
3. System components Magaprime DNA RPN1604 RPN1605 RPN1606 RPN1607 labelling Primer solution: 150 µl 300 µl 150 µl 300 µl Random nonamer primers in an aqueous solution Labelling buffer ; – – 300 µl 600 µl dA TP , dGTP and dT TP in Tris/HCl pH7.
Magaprime DNA RPN1604 RPN1605 RPN1606 RPN1607 labelling Enzyme solution; 60 µl 120 µl 60 µl 120 µl 1 unit/µl DNA polymerase 1 Klenow fragment (cloned in 100 mM potassium phosphate pH6.5, 10 mM 2-mercapto- ethanol and 50% glycerol Standard DNA 25 µl 50 µl 25 µl 50 µl solution; 5 ng/µl Hin d III digested lambda DNA in 10 mM Tris/HCl pH 8.
3.1. Megaprime DNA labelling systems 30 standard labelling reactions – for use with any radioactive nucleotide RPN 1604 60 standard labelling reactions – for use with any radioactive nucleotide RP.
4. Introduction Feinbereg and Vogelstein (1,2) intr oduced the use of random sequence hexancleotides to prime DNA synthesis on denatured template DNA at numerous sites along its length. The primer- template complex is a substrate for the ‘Klenow’ fragment of DNA polymerase 1.
10 Figure 1. Preparation of labelled pr obes using GE Healthcare’s megaprime DNA labelling systems. Linear dsDNA Denature in presence of monamer primers Add Multiprime DNA reaction buffer Add labelled dNTP and ‘Klenow’ DNA polymerase.
11 Protocol 1. Dissolve the DNA to be labelled to a concentration of 2.5–25 ng/µl in either distilled water of 10 mM Tris/HCl, pH8.0, 1 mM EDT A (TE buffer). Notes 1. If desired, the labelling efficiency of a DNA sample can be compared with that of the standard DNA supplied with the kit .
12 Protocol 2. Place the required tubes fr om the Megaprime system, with the exception of the enzyme, at room temperature to thaw . Leave the enzyme at -15°C to -30°C until required, and return immediately after use.
13 Protocol Component RPN1604/5 RPN1606/7 Labelling 10 µl buffer Unlabelled 4 µl of each – dNTPs omitting those to be used as label Reaction 5 µl – buffer Radiolabelled (dNTP) 5 µl 5 µl (dCTP) Enzyme 2 µl 2 µl 6. Mix gently by pipetting up and down and cap the tube.
14 Protocol 7. Incubate at 37°C for 10 minutes continued. 8. Stop the reaction by the addition of 5 µl of 0.2 M EDT A . For use in a hybridization, denature the labelled DNA by heating to 95–100°C for 5 minutes, then chill on ice. Notes 7. Continued.
15 Protocol 8. Stop the reaction by the addition of 5 µl of 0.2 M EDT A . For use in a hybridization, denature the labelled DNA by heating to 95–100°C for 5 minutes, then chill on ice continued. Notes 8. Continued under the conditions given above is not requir ed with the isotopes 32 P and 33 P.
16 Protocol 3. Place 25 ng (5 µl) of template DNA into a clean microcentrifuge tube and to it add 5 µl of primers. Denature by heating to 95–100°C for 5 minutes in a boiling water bath. 4. Spin briefly in a microcentrifuge to bring the contents to the bottom of the tube.
17 Protocol Reaction 5 µl – buffer Enzyme 2 µl 2 µl Water* as appropriate for a final reaction volume of 50 µl* * When calculating this volume remember to allow for the volume of radioactive nucleotide to be added. 6. Cap the tube and spin for a few seconds in a microcentrifuge to bring the contents to the bottom of the tube.
18 Protocol 8. Incubate at 37°C for 10 minutes continued. 9. Stop the reaction by the addition of 5 µl of 0.2 M EDT A . For use in a hybridization, denature the labelled DNA by heating to 95–100°C for 5 minutes, then chill on ice.
19 Protocol 9. Stop the reaction by the addition of 5 µl of 0.2 M EDT A . For use in a hybridization, denature the labelled DNA by heating to 95-100°C for 5 minutes, then chill on ice continued . Notes 9. Continued described in Appendix III. Calculation of probe specific activity is described in Appendix II.
5.3. Use of alternative reaction conditions a. Use of more than one labelled [ α – 32 P]dNTP . Table 1 lists the r esults of a selection of standard reactions, using a variety of input labels under optimum conditions. Figure 3 gives more complete information on their use in Megaprime reactions.
c. Use of [ 32 P]dNTP α S. When using 32 S-labelled radionucleotides the incubation time should be extended to 1 hour at 37°C. d. Labelling at room temperature. If desired, labelling reactions can be carried out at r oom temperature. Maximum incorporation occurs after an incubation time of 45–60 minutes.
22 a. At the specific activity reference date of the labelled nucleotide. b. Formulation code 1 = 370 MBq/ml, 10 mCi/ml in stabilized aqueous solution. c. The probe specific activities were calculated using observed incorporation levels which are similar to those found in figure 3b.
a) Specific activity i) One labelled dNTP ii) Two labelled dNTP iii) Three labelled dNTP Figure 3. The use of [ α – 32 P]dNTPs in the Megaprime DNA labelling system (see notes on page 26).
b) Incorporation efficiency i) One labelled dNTP ii) Two labelled dNTP iii) Three labelled dNTP Figure 3. The use of [ α – 32 P]dNTPs in the Megaprime DNA labelling system (see notes on page 26).
25 c) Probe length i) One labelled dNTP ii) Two labelled dNTP iii) Three labelled dNTP Figure 3. The use of [ α – 32 P]dNTPs in the Megaprime DNA labelling system (see below). Notes to figure 3 a. The results shown are the means of a number of experiments in which different nucleotides and combinations of nucleotides were used.
c. The data was generated using the standard labelling pr otocols. If dNTPs <3000 Ci/mmol are to be used, then the desired pr obe specific activity must be multiplied by a conversion factor , before determining the amount of input label.
27 Protocol 1. Fractionate restriction endonuclease digested DNA in a suitable low melting point agarose gel containing 0.5 µg/ml ethidium bromide. Estimate the DNA content of the band by reference to a set of standards of known concentration on another track.
28 Protocol 3. Add water to a ratio of 3 ml per gram of gel and place in a boiling water bath for 5 minutes to melt the gel and denature the DNA. 4. If the DNA is to be used immediately remove the appropriate volume containing 25 ng, add to the primers as indicated in the labelling protocol (page 11, step 3).
29 Protocol 1. Remove a 1 or 2 µl aliquot of the reaction mixture to a clean microcentrifuge tube containing 20 µl of water or 10 mM Tris/HCl pH.8.0.
30 Protocol 5. Place the squares in separate vials with at least 5 ml of scintillation fluid and count . 6. Efficiency of counting will vary, but the percentage incorporation can be used to calculate probe specific activity.
31 Protocol 6. Continued. The amount of radioactivity incorporated during the reaction (B) in dpm. B = total number of µCi added x 2.2x10 4 x % incorporation Thus the specific activity of the labelled DNA is specific activity = B x 10 3 dpm per µg specific activity = A Notes B.
6. Wash the filter discs six times with 2 ml 10% TCA solution and dry the filter discs thoroughly, for example using an infra-red lamp. Avoid overheating and possible charring of the discs. 7. Count the dried filter discs by liquid scintillation or Cerenkov ( 32 P) and count with the samples set aside in step 3.
any liquid from the microcentrif uge tube. Refill with Sephadex and centrifuge as before. Continue until the column is packed to a volume of 1 ml. ™ Sephadex is a trademark of GE Healthcare 4. Add a volume of TE buffer equal to the reaction volume, to the top of the column and centrifuge, as in step 3.
6. Wash the pellet once in 90% ethanol, in the same manner . Dry the pellet . 7. Finally redissolve the DNA pellet in TE buffer for use as a pr obe and for storage. 6.4. Appendix IV . Additional equipment and reagents TE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDT A) 0.
35 Problem 1. Low signal Possible cause 1. Incomplete denaturation of template DNA 2. Low probe concentration 3. Low probe specific activity Remedy 1. Ensure denaturation protocol is followed. 2. Accurately measure the concentration of template DNA used in the labelling reactions.
36 Problem 2. Non-specific background over whole of filter Possible cause 4. Loss of dNTP during evaporation 1. Presence of unincorporated label Remedy 4. If the dNTP solution has been evaporated to dryness prior to use, handling losses may have occurred.
37 Problem Possible cause 2. Concentrated probe has contacted membrane directly during probe addition 3. Probe concentration is too high 4. Probe not denatured Remedy 2. It is suggested that up to 1.0 ml of the buffer used for prehybridization is withdrawn for mixing with the probe.
8. Refer ences 1. FEINBERG, A .P . and VOGELSTEIN, B., Anal. Biochem. , 132 , pp.6-13, 1983. 2. FEINBERG, A .P . and VOGELSTEIN, B., Addendum Anal. Biochem., 137 , pp.266-267, 1984. 3. SOUTHERN, E.M., J.Mol.Biol., 98 , pp.503-517, 1975. 4. THOMAS, P .
9. Related Pr oducts Labelling systems Nick translation kits N5000/5500 3’-end labelling kit N4020 5’ end labelling kit RPN 1509 RNA labelling system (paired promoter SP6/T7 system) RPN 3100 Hybri.
Table 2. Labelled dNTPs and analogues available from GE Healthcar e Compound Specific Activity Formulation Product TBq/mmol Ci/mmol (see key) code [ α – 32 P]dA TP ~220 ~6000 1 PB 1074 ~110 ~3000 1.
Compound Specific Activity Formulation Product TBq/mmol Ci/mmol (see key) code [ 35 S]dCTP α S >37 >1000 1 SJ1305 ~22 ~600 1 SJ 305 ~15 ~400 1 SJ 265 [ 35 S]dGTP α S ~22 ~600 1 SJ 306 [ 35 S]dT TP α S ~22 ~600 1 SJ 307 [8– 3 H]dA TP 0.37–1.
imagination at work RPN1604PL Rev B 2006 http://www . gehealthcare.com/lifesciences GE Healthcare UK Limited Amersham Place, Little Chalfont , Buckinghamshire, HP7 9NA UK GE Healthcare regional office.
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