GE RPN1607 manuel d'utilisation

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. Standard megaprime pr otocol 11 5.2. New megaprime protocol 15 5.3. Use of alternative reaction[...]

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. General Electric Company reserves the right , subject to any regulatory and c[...]

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. This product is to be used with radioactive material. Please follow the manufacturer ’s[...]

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). Incorporations greater than 55% are achiev ed after 10 minutes incubation at 37°C, as assayed by thin- layer chromatography on[...]

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 pH7.5, 2-mercaptoethanol and MgCl 2 Nucleotide solutions (a) dA TP 120 µl 240 µl – – (b) cCTP [...]

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 8.0,[...]

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 RPN 1605 30 standard labelling reactions – for use with radioactively labelled dCTP RPN 1606 60 standard labelling reactions – for use with radioact[...]

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. By substituting a radiolabelled nucleotide for a non- radioactive equ[...]

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. Incubate Denature to release labelled probe and add directly to hybridization Random sequence monamers Unabel[...]

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 . In this case 5 µl of standard DNA should be used. 5. Meg[...]

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. 3. Place 25 ng of template DNA into a microcentrifuge tube and to it add 5 µl of primers and the appropriate volume of water to gi[...]

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. Spin for a few seconds in a microcentrifuge to bring the contents to the[...]

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. times (up to 60 minutes) are requir ed when nucleotide analogues (e.g. [ 35 S]dNTP α S) are u[...]

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. Purification of 35 S labelled probes is however r[...]

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. 5. Keeping the tube at room temperature add the nucleotides and 10x react[...]

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. 7. Add the radio[...]

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. Notes 8. Continued When labelling DNA in low melting point agarose, longer incubation of 15–30 minutes at 3 7°C[...]

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. Extensive experimentation with Rapid-h[...]

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. Reactions were carried out at 37°C for 5 minutes. [...]

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. A decline in incorporation can be observ[...]

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. d. It is impor tant to note that the specific activity of probes m[...]

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). 23 Specific activity of the labelled product (dpm/µg) x 10 9 5 4 3 2 1 (iii) (ii) (i) 0 1 0 20 30 40 5 0 60 70 80 9 0 10 0 Total input label (pmols)[...]

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). 24 Percentage of added label 100 80 60 40 20 (i) (ii) (iii) 0 1 0 20 30 40 5 0 60 70 80 9 0 10 0 Total input label (pmols)[...]

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. Observed results [...]

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. For a single labelled dNTP:- Total input label (pmols) = 3000 Ci/mmol x required probe specific activity of s[...]

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. 250 ng should allow 25 ng to be used in the standard labelling protocol without further [...]

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). The volume of DNA should not exceed 25 [...]

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. 1 mM EDT A buffer . Mix well by pipetting up and down. 2. Spot , in quadruplicate, 5 µl aliquots of this dilution on to Whatman DE81 chromatography paper squares (minimum size 1 x 1 cm), placed on [...]

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. Unlike the nick translation labelling reaction, Megaprime labelling leads to net DNA synthesis, and so the total amount of DN[...]

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. Precipitation with trichloroacetic acid Plastic or siliconized glass [...]

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. 8. Determine % incorporation and pro[...]

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. A minimum of 50 µl should be applied to the[...]

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) 0.2 M EDT A solution Adjustable pipettes for example Pipetman™ Sterile pipette tips[...]

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. Check recovery of probe if purification is performed to remove uninc[...]

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. Check this loss has not occurred during lyophilization of the solvent , during transfer of the[...]

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. The mixture should then be added back to the hybridization contain[...]

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 . S., Proc . Natl. Acad . Sci. US A ., 77 , pp.5201-5205, 1980. 5. MEINKO TH, J. and W AHL , G., Ana[...]

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 Hybridization buffers Rapid-hyb buffer RPN 1635/6 Hybridization buffer tablets RPN 131 Hybridization membranes Hybond™ - Range of nylon and nitrocellulos[...]

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 PB 10204 2 PB 204 ~30 ~800 1 PB 10384 ~15 ~400 1 PB 10164 2 PB 164 [ α – 32 P]dCTP ~220 ~6000 1 PB 10475 ~110 ~3000 2 PB 10205 1 PB 205 ~30 ~6000 [...]

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 0.37–1.1 10–30 2 TRK 347 [1’ ,2’ ,2,8– 3 H]dA TP 1.83–3.7 50–100 2 TRK 633 [1’ ,2’ ,5–[...]

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 contact numbers: Asia Pacific Tel: +85 65 62751830 Fax: +85 65 62751829 Australasia Tel: + 61 2 8820 8299 Fax: +61 2 8820 8200 Austria Tel: 01/57606-[...]