GE Healthcare Amersham Megaprime™ DNA Labelling Systems Product Booklet Codes: RPN1604 RPN1605 RPN1606 RPN1607
Page finder 1. Legal 3 2. Handling 2.1. Safety warnings and precautions 2.2. Storage and stability 2.3. Quality control 4 4 4 4 3. System components 3.1. Megaprime DNA labelling systems 6 8 4. Introduction 9 5. Megaprime DNA labelling protocols 5.1. Standard megaprime protocol 5.2. New megaprime protocol 5.3. Use of alternative reaction conditions 11 11 15 20 6. Appendices 6.1. Appendix I. Labelling of DNA fragments in low melting point agarose 6.2. Appendix II.
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.
2. Handling 2.1. Safety warnings and precautions safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes, wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice. Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. 2.2.
using 17 pmol/25 ng DNA of [α–32P] 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–32P]dCTP, 3000 Ci/mmol (code PB 10205). Incorporations greater than 55% are achieved after 10 minutes incubation at 37°C, as assayed by thinlayer chromatography on PEI cellulose in 1.25 M KH2PO4. PH3.4. In addition components of the kits are checked for identity by HPLC and the DNA solutions for concentration by UV spectrophotometry.
3. System components Magaprime DNA labelling RPN1604 RPN1605 RPN1606 RPN1607 Primer solution: Random nonamer primers in an aqueous solution 150 µl 300 µl 150 µl 300 µl Labelling buffer; dATP, dGTP and dTTP in Tris/HCl pH7.5, 2-mercaptoethanol and MgCl2 – – 300 µl 600 µl (a) dATP (b) cCTP (c) dGTP (d) dTTP in Tris/HCl pH8.0, 0.5 mM EDTA 120 µl 120 µl 120 µl 120 µl 240 µl 240 µl 240 µl 240 µl – – – – – – – – Reaction buffer: A 10x concentrated buffer containing Tris/HCl pH7.
Magaprime DNA labelling RPN1604 RPN1605 RPN1606 RPN1607 Enzyme solution; 60 µl 1 unit/µl DNA polymerase 1 Klenow fragment (cloned in 100 mM potassium phosphate pH6.5, 10 mM 2-mercaptoethanol and 50% glycerol 120 µl 60 µl 120 µl Standard DNA solution; 5 ng/µl Hind III digested lambda DNA in 10 mM Tris/HCl pH 8.0, 1 mM EDTA 25 µl 50 µl 25 µl 50 µl Carrier DNA solution; 500ng/ml sonicated herring sperm DNA in 10 mM Tris/HCl pH 8.0, 1 mM EDTA 1.25 ml 2.5 ml 1.25 ml 2.
3.1.
4. Introduction Feinbereg and Vogelstein (1,2) introduced the use of random sequence hexancleotides to prime DNA synthesis on denatured template DNA at numerous sites along its length. The primertemplate complex is a substrate for the ‘Klenow’ fragment of DNA polymerase 1. By substituting a radiolabelled nucleotide for a nonradioactive equivalent in the reaction mixture newly synthesized DNA is made radioactive (see Figure 1).
Labelled dNTP ‘Klenow’ polymerase Unabelled dNTPs Random sequence monamers Figure 1. Preparation of labelled probes using GE Healthcare’s megaprime DNA labelling systems. Denature to release labelled probe and add directly to hybridization Add labelled dNTP and ‘Klenow’ DNA polymerase.
5. Megaprime DNA labelling protocols The Megaprime systems allow DNA from a variety of sources to be labelled in vitro to high specific activity with 32P and other radionuclides. The specific activity of the probes generated by these systems will vary according to the specific activity of the labelled dNTP used. The standard Megaprime protocol is presented, together with a new protocol which reduces the variation in labelling efficiency that can occur with DNA template from a variety of sources.
Protocol Notes 2. Place the required tubes from 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 give a total volume of 50 µl in the final Megaprime reaction. Denature by heating to 95–100°C for 5 minutes in a boiling water bath. 3.
Protocol Component Notes RPN1604/5 Labelling RPN1606/7 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 bottom of the tube. 6. Avoid vigorous mixing of the reaction mixture as this can cause severe loss of enzyme activity. 7.
Protocol Notes 7. Incubate at 37°C for 10 minutes continued. 7. Continued. times (up to 60 minutes) are required when nucleotide analogues (e.g. [35S]dNTPαS) are used. 8. Stop the reaction by the addition of 5 µl of 0.2 M EDTA. For use in a hybridization, denature the labelled DNA by heating to 95–100°C for 5 minutes, then chill on ice. 8. Labelled probe can be stored at -15°C to -30°C in a non frost-free freezer. Prolonged storage of 32P-labelled probes can lead to substantial probe degradation(7).
Protocol Notes 8. Stop the reaction by the addition of 5 µl of 0.2 M EDTA. For use in a hybridization, denature the labelled DNA by heating to 95–100°C for 5 minutes, then chill on ice continued. 8. Continued under the conditions given above is not required with the isotopes 32P and 33P. Purification of 35S labelled probes is however required to reduce filter background. 5.2. New Megaprime protocol Protocol Notes 1.
Protocol Notes 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. 3. If the volume of DNA and primers is less than 10 µl make up to this volume with water. When labelling DNA in low melting point agarose first place the tube containing the stock DNA in a boiling water bath for 30 seconds to melt the agarose before removing the required volume.
Protocol Reaction buffer Notes 5 µl – 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 radiolabelled dNTP, for example 5µl [α–32P]dNTP, specific activity 3000 Ci/mmol. Mix by gently pipetting up and down.
Protocol Notes 8. Incubate at 37°C for 10 minutes continued. 8. Continued When labelling DNA in low melting point agarose, longer incubation of 15–30 minutes at 37°C are required for optimum labelling. Longer incubation times (up to 60 minutes) are required when nucleotide analogues (e.g. [35S]dNTP(S) are used. 9. Stop the reaction by the addition of 5 µl of 0.2 M EDTA. For use in a hybridization, denature the labelled DNA by heating to 95–100°C for 5 minutes, then chill on ice. 9.
Protocol Notes 9. Stop the reaction by the addition of 5 µl of 0.2 M EDTA. For use in a hybridization, denature the labelled DNA by heating to 95-100°C for 5 minutes, then chill on ice continued. 9. Continued described in Appendix III. Calculation of probe specific activity is described in Appendix II. Extensive experimentation with Rapid-hyb buffer (RPN1635/6) has shown that probe purification, even under the conditions given above is not required with the isotopes 32P and 32P.
% added label incorporated into DNA 90 80 70 60 50 40 30 20 10 0 10 20 30 40 50 60 Length of incubation in minutes Figure 2. Time course of incorporation of [α–32P]dCTP (17 pmoles) in a Megaprime reaction at 37°C. The DNA used was the standard DNA supplied with the system. 5.3. Use of alternative reaction conditions a. Use of more than one labelled [α–32P]dNTP. Table 1 lists the results of a selection of standard reactions, using a variety of input labels under optimum conditions.
c. Use of [32P]dNTPαS. When using 32S-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 room temperature. Maximum incorporation occurs after an incubation time of 45–60 minutes. A decline in incorporation can be observed if reactions are left overnight. e. Factors affecting the labelled DNA. 1.
5.3 x 109 1.9 x 109 Quantity of each dNTP required 32 } 3.4 x 10 Product code 200 20 17 Formulation (see note b) 5 17 17 Specific activity (see note a) 7.4 50 5 5 Compounds PB 10205 1.85 50 50 dpm/ug PB 10475 1.85 Specific activity of probe (see notes c,d and e) 1 PB 10204 1.85 17 Ci/mmol 1 PB 10205 pmol ~3000 1 PB 10204 17 17 5 ~6000 1 5 5 µl ~111 TBq/mmol ~3000 1 50 50 50 ~222 ~3000 1.85 1.85 µCi ~111 ~3000 PB 10206 PB 10205 1.
Specific activity of the labelled product (dpm/µg) x 109 a) Specific activity 5 (iii) 4 (ii) 3 (i) 2 1 0 10 20 30 40 50 60 70 80 90 100 Total input label (pmols) i) One labelled dNTP ii) Two labelled dNTP iii) Three labelled dNTP Figure 3. The use of [α–32P]dNTPs in the Megaprime DNA labelling system (see notes on page 26).
Percentage of added label b) Incorporation efficiency 100 80 60 (i) (ii) (iii) 40 20 0 10 20 30 40 50 60 70 80 90 100 Total input label (pmols) i) One labelled dNTP ii) Two labelled dNTP iii) Three labelled dNTP Figure 3. The use of [α–32P]dNTPs in the Megaprime DNA labelling system (see notes on page 26).
Mean probe length (bases) c) Probe length 100 80 60 (i) (ii) (iii) 40 20 0 10 20 30 40 50 60 70 80 90 100 Total input label (pmols) i) One labelled dNTP ii) Two labelled dNTP iii) Three labelled dNTP Figure 3. The use of [α–32P]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 may deviate ± 10% from those shown.
c. The data was generated using the standard labelling protocols. If dNTPs <3000 Ci/mmol are to be used, then the desired probe 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 specific activity dNTP to be used For more than one labelled dNTP the mean specific activity of the labelled dNTP to be used should be inserted in the above calculation.
6. Appendices 6.1. Appendix I. Labelling of DNA fragments in low melting point agarose The DNA samples produced by the following protocol have been found to be labelled to approximately the same extent as purified DNA. 15–20 minutes at 37°C is optimum for labelling. The standard labelling protocol may be found to be more appropriate for labelling DNA in agarose as the volume of DNA to be added using the new protocol is limited to 5 µl, requiring a relatively high initial DNA concentration.
Protocol Notes 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. 3. If the DNA is not to be used immediately divide the boiled samples into suitably sized aliquots and store at -15°C to -30°C in a non frost-free freezer. 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).
Protocol Notes 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 EDTA 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 a nonabsorbent backing. These squares may be marked with a pencil for identification if required. 3. Take two of the filters and dry under a heat lamp.
Protocol Notes 5. Place the squares in separate vials with at least 5 ml of scintillation fluid and count. 5. Determination of the proportion of the 32P labelled nucleotide incorporated may be achieved using Cerenkov counting if desired in this case drying the filter is not necessary. 6. Efficiency of counting will vary, but the percentage incorporation can be used to calculate probe specific activity.
Protocol Notes 6. Continued. The amount of radioactivity incorporated during the reaction (B) in dpm. B = total number of µCi added x 2.2x104 x % incorporation Thus the specific activity of the labelled DNA is specific activity = B x 103 dpm per µg specific activity = A B. Precipitation with trichloroacetic acid Plastic or siliconized glass tubes must be used to avoid adsorption of DNA. 1. Dilute an appropriate aliquot of the reaction mixture as described in section A1. 2.
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 (32P) and count with the samples set aside in step 3. 8. Determine % incorporation and probe specific activity as in section A6. 6.3. Appendix III.
any liquid from the microcentrifuge 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 column. 5. Repeat once more to ensure fractions of the correct size are collected from the column. 6.
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 probe and for storage. 6.4. Appendix IV. Additional equipment and reagents TE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA) 0.
7. Troubleshooting guide If poor results are obtained, the following guide may help to determine the cause of the problem. Problem Possible cause Remedy 1. Low signal 1. Incomplete denaturation of template DNA 1. Ensure denaturation protocol is followed. 2. Low probe concentration 2. Accurately measure the concentration of template DNA used in the labelling reactions. Check recovery of probe if purification is performed to remove unincorporated nucleotide. 3. Low probe specific 3.
Problem 2. Non-specific background over whole of filter Possible cause Remedy 4. Loss of dNTP during evaporation 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 reconstituted dNTP solution or by adsorption of the dNTP onto the walls of the tube.
Problem Possible cause Remedy 2. Concentrated 2. It is suggested probe has contacted that up to 1.0 ml membrane of the buffer used directly during for prehybridization probe addition is withdrawn for mixing with the probe. The mixture should then be added back to the hybridization container in an area away from the filter. 3. Probe concentration 3. Ensure measurement of template DNA is too high concentration is accurate 4. Probe not denatured 37 4.
8. References 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. USA., 77, pp.5201-5205, 1980. 5. MEINKOTH, J. and WAHL, G., Anal, Biochem., 138, pp. 267-284, 1984. 6. GRUNSTEIN M. and HOGNESS, D.S., Proc. Natl. Acad. Sci. USA., 72, pp. 3961-3965, 1975. 7. HODGSON, C.P., FISK, R.Z. and WILLET, L.B.
9.
Table 2.
Compound Specific Activity TBq/mmol Ci/mmol Formulation (see key) Product code [35S]dCTPαS >37 ~22 ~15 >1000 ~600 ~400 1 1 1 SJ1305 SJ 305 SJ 265 [35S]dGTPαS ~22 ~600 1 SJ 306 [35S]dTTPαS ~22 ~600 1 SJ 307 0.37–1.1 10–30 [8–3H]dATP [1’,2’,2,8–3H]dATP 1.83–3.7 50–100 [1’,2’,5–3H]dCTP 1.85–3.14 50–85 [5–3H]dCTP 0.55–1.1 15–30 [8–3H]dGTP 0.185–0.740 5–20 [1’2,(–3H]dGTP 0.9–1.85 25–50 [methyl, 1’,2’–3H]TTP 3.3–4.
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