GE RPN1605 User's Manual
Contents
1. 1 2 5 3H dCTP 1 85 3 14 50 85 2 TRK 625 5 3HJdCTP 0 55 1 1 15 30 2 TRK 352 8 5H dGTP 0 185 0 740 5 20 2 TRK 350 1 2 5H dGTP 0 9 1 85 25 50 2 TRK 627 methyl 1 2 3H TTP 3 3 4 8 90 130 2 TRK 576 methyl 3HITTP 40 60 2 TRK 424 30 2 TRK 354 125IJdCTP gt 55 gt 1500 3 IM 5103 Formulation codes 1 370 MBq ml 10 mCi ml in stabilized solution 2 37 MBq ml 1 mCi ml in 50 aqueous ethanol 3 185 MBq ml 5 mCi ml in 50 aqueous ethanol See GE Healthcare Products catalogue for further details 41 GE Healthcare offices GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue PO Box 1327 Piscatawau NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan http www gehealthcare com lifesciences 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 1613 Fax 01 57606 1614 Belgium Tel 0800 73 890 Fax 02 416 8206 Canada Tel 1 800 463 5800 Fax 1 800 567 1008 Central East amp South East Europe Tel 43 1972 720 Fax 43 1 972 722 750 Denmark Tel 45 70 25 22. High specific activity probes should be stored for no longer than 3 days Although probe purification is not usually necessary for most membrane applications he removal of unicorporated nucleotide is sometimes useful to reduce background in filter hybridizations or probes 10 dpm ug or when the reaction yields an incorporation of less than 5096 This procedure is 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 Protocol 8 Stop the reaction by the addition of 5 ul 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 Notes 8 Continued under the conditions given above is not required with the isotopes P and P Purification of 35S labelled probes is however required to reduce filter background 5 2 New Megaprime protocol Protocol l Dilute the DNA to a concentration of 5 ng pl in either distilled water or 10 mM TE buffer 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 Notes 1 DNA solutions at concentrations in the range 5 25 ng ul can be used if desired However the denaturing volume step 3 sho
3. 10 mM 2 mercapto ethanol and 50 glycerol Standard DNA solution 5 ng ul Hind III digested lambda DNA in 10 mM Tris HCl pH 8 0 1 mM EDTA 25 ul Carrier DNA solution 500ng ml sonicated herring sperm DNA in 10 mM Tris HCI pH 8 0 1 mM EDTA 1 25 ml RPN1605 RPN1606 120 ul 60 ul 50 ul 25 ul 2 5 ml 1 25 ml RPN1607 120 ul 50 ul 2 5 ml 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 radioactively labelled dCTP RPN 1607 4 Introduction Feinbereg and Vogelstein 1 2 introduced the use of random sequence hexancleotides to prime DNA synthesis on denatured emplate DNA at numerous sites along its length The primer emplate complex is a substrate for the Klenow fragment of DNA polymerase 1 By substituting a radiolabelled nucleotide for a non radioactive equivalent in the reaction mixture newly synthesized DNA is made radioactive see Figure 1 The absence of the 5 3 exonuclease activity associated with DNA polymerase 1 ensures hat labelled nucleotides incorporated by the polymerase are not subsequently removed as monophosphates Very small amount of input DNA can be labelled enabling very high specific
4. Spin in a microcentrifuge for 15 minutes Carefully aspirate and dispose of supernatant in a suitable manner 5 Wash the pellet once in 0 5 ml of 0 67 M ammonium acetate pH 4 5 6796 ethanol at room temperature by gentle inversion centrifugation and aspiration 33 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 HCI pH 8 0 1 mM EDTA 0 2 M EDTA solution Adjustable pipettes for example Pipetman Sterile pipette tips Waterbaths at 37 C and 100 C Polypropylene microcentrifuge tubes icrocentrifuge Gloves Radiation safety equipment DE81 ion exchange chromatography paper Whatman Trichloroacetic acid TCA solution 10 w v TCA in water Filter discs glass fibre or nitrocellulose Plastic or siliconized glass tubes capacity 5 ml Filtration apparatus 2x SSC 30 mM Na citrate 300 mM NaCl pH 7 0 Pipetman is a registered trademark of Gilson 34 7 Troubleshooting guide If poor results are obtained the following guide may help to determine the cause of the problem Problem 1 Low signal Possible cause 1 Incomplete denaturation of template DNA 2 Low probe concentration Remedy 1 Ensure denaturation protocol is followed 2 Accurately measure the concentration of template DNA used in the labell
5. activity DNA probes to be produced with relatively small quantities of added nucleotides These radioactive labelled fragments can then be used as sensitive hybridization probes for a wide range of filter based applications 3 6 Previous protocols for the random primer labelling of DNA have required reaction times of at least 30 minutes GE Healthcare s agaprime DNA labelling system allows the labelling of template DNA to the same high specific activity but at a greatly accelerated rate Probes of specific activity 1 9x10 dpm ug can be produced with he majority of DNA substrates using the standard protocol after 10 minutes incubation at 37 C This rapid labelling is achieved by he use of nonamer primers rather than the conventional hexamers Figure 1 Nonamers allow for more efficient priming from the emplate DNA at 37 C resulting in fast and efficient labelling of the DNA A new alternative protocol has further reduced the variability in labelling which can occur with DNA template from a variety of sources Both the standard Megaprime protocol and the new protocol are given as options in this booklet The labelling of DNA in ow melting point agarose takes only 15 30 minutes in contrast to conventional systems where overnight incubation are necessary sujejsfis Guijjadp YNG euuudpb6au s D dINP P9II99D1 m 0 espJeuufijod moua y a 9 CEXTIITTIIITIIOND CEEITTIIIITNMEN SdINP pallegoun o SJoUJDUOUJ
6. area away from the filter 3 Probe concentration 3 Ensure measurement is too high of template DNA concentration is accurate 4 Probe not denatured 4 Non denatured double stranded probes often give high backgrounds 37 8 References FEINBERG A P and VOGELSTEIN B Anal Biochem 132 pp 6 13 1983 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 HODGSON C P FISK R Z and WILLET L B Biotechniques 6 pp 208 211 8 SAMBROOK J FRITSCH E F and MANIATIS T Molecular Cloning a laboratory manual second edition Cold Spring Harbour Laboratory 1989 9 MUNDY C R CUNNINGHAM M W and READ C A Essential Molecular Biology Practical Approach Vol 2 T A Brown ed Oxford University Press Oxford 1991 pp 57 109 E N N 38 9 Related Products Labelling systems ick 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 nitrocellul
7. be stored for no longer than 3 days Although probe purification is not usually necessary for most membrane applications the removal of unincorporated nucleotide is sometimes useful to reduce background in filter hybridizations for probes 10 dpm ug or when the reaction yields an incorporation of less than 5096 This procedure is 18 Protocol Notes 9 Continued 9 Stop the reaction by the addition of 5 ul 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 19 described in Appendix III Calculation of probe specific activity is described in Appendix Il 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 P and P Purification of 32S labelled probes is however required to reduce filter background 90 2 80 3 gt c x T 70 oO o 5 60 e g 50 g 40 Es 3 30 e 20 10 0 10 20 30 40 S0 60 Length of incubation in minutes Figure 2 Time course of incorporation of o 5 P dCTP 17 pmoles in a Megaprime reaction at 37 C The DNA used was the standard DNA supplied with the system 5 5 Use of alternative reaction conditions a Use of more than one labelled o 5 P dNTP Table 1 lists the results of a selection of standard reactions using a variety of input labels under optimum conditi
8. 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 52P and count with the samples set aside in step 3 8 Determine 96 incorporation and probe specific activity as in section A6 6 3 Appendix IIl Removal of unincorporated nucleotides Removal of unincorporated nucleotides is sometimes desirable to reduce background produced by the probe during hybridization It is considered important to remove these free nucleotides particularly if the radioactive probe is to be kept for several days before use or the incorporation is less than 50 If 5 P or 33P labelled probes are to be used in combination with GE Healthcare s new Rapid hub buffer RPN1635 6 purification is not required unless the probe is to be used more than 24 hours after preparation Probes can be purified by Sephadex chromatography or selective precipitation 8 9 A Sephadex G 50 spin columns Probe reaction are passed through columns packed with Sephadex G 50 which retains the free nucleotides within the column matrix A number of pre packed columns are commercially available However columns may also be prepared as indicated below 1 Equilibrate Sephadex G 50 in TE buffer either overnight or at 65 C for 1 2 hours 2 Plug a 1 0 ml syringe with a piece of siliconized glass wool 3 Fill the syringe with the equilibrated Sephadex Place in a 15 ml conical tube
9. the reaction mix such that incorporation mean counts on washed filters x 100 mean counts on unwashed filters Protocol Notes 6 Continued The amount of radioactivity incorporated during the reaction B in dpm B total number of uCi added x 2 2x104 x 96 incorporation Thus the specific activity of the labelled DNA is specific activity B x 105 dpm per pg 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 N Transfer 1 10 ul of diluted reaction mixture to two duplicate tubes containing 200 ul water or 0 2M EDTA and 50 ul carrier DNA solution Mix well Use this mixture less any set aside in step 3 for the TCA precipitation described in step 4 below 3 Set aside an appropriate aliquot from each tube in step 2 for the determination of total input radioactivity 4 To the diluted samples from step 2 add 2 ml ice cold 10 trichloroacetic acid TCA solution vortex and allow to stand in an ice bath for 10 15 minutes The labelled and carrier DNA will co precipitate Note that TCA is corrosive and care should be taken in its handling en Collect the precipitated DNA by vacuum filtration on a glass fibre or nitrocellulose filter disc 31 6 Wash the filter discs six times with 2 ml 10 TCA solution and dry the filter discs thoroughly for
10. 4 50 Fax 45 45 16 2424 Eire Tel 1 800 709992 Fax 44 1494 542010 Finland amp Baltics Tel 4358 9 512 3940 Fox 358 9 512 39439 GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK France Tel 01 69 35 67 00 Fax 01 69 41 98 77 Germany Tel 0800 9080 711 Fax 0800 9080 712 Greater China Tel 852 2100 6300 Fax 852 2100 6338 Italy Tel 02 26001 320 Fax 02 26001 399 Japan Tel 4813 53319336 Fax 481 3 53319370 Korea Tel 82 2 6201 3700 Fax 82 2 6201 3803 Latin America Tel 55 11 3933 7300 Fax 55 11 3933 7304 Middle East amp Africa Tel 30 210 96 00 687 Fax 30 210 96 00 693 Netherlands Tel 0800 82 82 82 1 Fax 0800 82 82 82 4 Norway Tel 47 815 65 777 Fax 47 815 65 666 imagination at work Portugal Tel 21 417 7035 Fax 21 417 3184 Russia C LS amp N I S Tel 7 495 956 5177 Fax 7 495 956 5176 Spain Tel 902 11 72 65 Fax 935 94 49 65 Sweden Tel 018 612 1900 Fax 018 612 1910 Switzerland Tel 0848 8028 10 Fax 0848 8028 11 UK Tel 0800 515 313 Fax 0800 616 927 USA Tel 1 800 526 3593 Fax 1 877 295 8102 RPN1604PL Rev B 2006
11. B aouanbas uopuby B a CEEEEZIIIT B ag ETETIITIT 31P9y P2H 39 Guisn seqoud paj eqp jo uonpupdaug T e1n614 uonpzipuqfiu oj fi eup ppo pup aqoud paj eqp 9SD8 31 0 eJnjbUue apqnou aspjawAjod VNG Moua y pub dINP Pelleqp ppv Jejjng uon2paiJ VNQ euuudnini ppy sjeuud JBUIDUOUI Jo aduasaud Ul eynjpue VNASP Daun 10 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 The protocols given here are for use with 17 pmolla32P dNTP specific activity 3000 Ci mmol For alternative reaction conditions refer to page 20 DNA prepared by standard minilusate methods may be used in either protocol DNA solutions which are too dilute to be used directly should be concentrated by ethanol precipitation followed by redissolution in an appropriate volume of water or 10 mM Tris HCI pH 8 0 1 mM EDTA DNA in restriction enzyme buffers may be added directly to the reaction The reaction can also be performed with DNA in agarose gel slices see note 3 and Appendix 1 5 1 Standard
12. GE Healthcare Amersham Megaprime DNA Labelling Systems Product Booklet Codes RPN1604 RPN1605 RPN1606 RPN1607 Page finder 1 Legal 2 Handling 2 1 Safety warnings and precautions 2 2 Storage and stability 2 3 Quality control 3 System components 3 1 Megaprime DNA labelling systems 4 Introduction 5 Megaprime DNA labelling protocols 5 1 Standard megaprime protocol 5 2 New megaprime protocol 5 3 Use of alternative reaction conditions 6 Appendices 6 1 Appendix Labelling of DNA fragments in low melting point agarose 6 2 Appendix II Monitoring the reaction and calculating the specific activity of the labelled DNA 6 3 Appendix III Removal of unincorporated nucleotides 6 4 Appendix IV Additional equipment and reagents 7 Troubleshooting guide 8 References 9 Related products ON FRADA U 11 15 20 27 27 28 32 34 35 38 39 1 Legal GE and GE monogram are trademarks of General Electric Companu 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 contractual approval if required to make changes in specifications and features shown herein or discontinue the product described at any time without notice or obligation Contact your GE Representative for the mo
13. Megaprime protocol Protocol Notes 1 Dissolve the DNA to be 1 If desired the labelling labelled to a concentration efficiency of a DNA sample of 2 5 25 ng ul in either can be compared with that distilled water of 10 mM of the standard DNA Tris HCl pH8 0 1 mM EDTA supplied with the kit In TE buffer this case 5 ul of standard DNA should be used 11 Protocol 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 ul of primers and the appropriate volume of water to give a total volume of 50 ul in the final Megaprime reaction 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 reaction buffer RPN 1604 5 or the labelling buffer RPN 1606 7 followed by the radiolabelled dNTP s and enzyme as follows 12 Notes 3 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 The volume of low melting point agarose DNA should not exceed 25 ul in a 50 ul reaction The reaction
14. ately divide the boiled samples into suitably sized aliquots and store at 15 C to 30 C in a non frost free freezer When using DNA which has been previously boiled and then stored at 15 C to 30 C first place the tube in a boiling water bath for 30 seconds to melt the agarose before removing the required volume containing 25 ng Do not reboil DNA aliquots more than twice 6 2 Appendix Il Monitoring the reaction and calculating the specific activity of the labelled DNA A Adsorption to DE81 paper Monitoring of the progress of the labelling reaction and measurement of probe specific activity can be achieved by determining the proportion of the radionucleotide incorporated during the Megaprime reaction 28 Protocol Notes 1 Remove a 1 or 2 ul aliquot of the reaction mixture to a clean microcentrifuge tube containing 20 ul 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 ul aliquots of this dilution on to Whatman DE81 chromatography paper squares minimum size 1x1 cm placed on a non absorbent 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 10 15 minutes should be adequate 4 Wash the remaining two 4 In aqueous solution DE81 filters twice for 5 minutes paper becomes fragile and each at room temperature care should be taken when in exce
15. he figure was obtained using linearized plasmid DNA 4 5 Kb in length under the standard labelling conditions It is recommended that not less than 10 pmol and not more than 125 pmol of any labelled dNTP is used in the reaction and combinations shown offer optimum balance of stability specific activity and economy 21 Compounds Specific activity Formulation Product Quantity of each Spe see note a see note b code dNTP required activity of probe see notes c d and e TBq mmol Ci mmol MBq pCi pl pmol dpm ug o 32P dCTP 111 3000 1 PB 10205 185 50 5 17 1 9 x 10 222 6000 1 PB 10475 74 200 20 32 5 3 x 10 a PJ dATP 111 3000 1 PB 10204 1 85 50 5 17 34x10 4x1 lo PJaCTP 11 3000 PB 10205 185 50 5 17 o 32P dATP 111 3000 1 PB 10204 1 85 50 5 17 o 32P dCTP 111 3000 1 PB 10205 1 85 50 5 17 3 7 x 10 lo P JdGTP 111 3000 1 PB 10206 185 50 5 17 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 important to note that the specific activity of probes made from different amounts of labelled dNTP cannot be calculated on a proportional basis because net DNA synthesis occurs e Brackets enclose nucleotides
16. iciency is reduced the labelled product is of a higher specific activity 25 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 Having determined the required number of pmols of input label with reference to figure 3a the required volume of each labelled dNTP can be calculated Note that the figures give the total amount of input label required If more than one labelled dNTP is to be used this figure should be divided by the number of labelled dNTPs to be used to give the required number of pmols of each labelled dNTP Volume of each labelled dNTP required in ul pmol of dNTP required x specific activity of dNTP Ci mmol x 10 3 radioactive concentration of dNTP mCi ml 26 6 Appendices 6 1 Appendix 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 p
17. in which a decapped 1 5 ml microcentrifuge tube has been inserted Centrifuge at 1600 g for 5 minutes Remove 32 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 ul should be applied to the column 5 Repeat once more to ensure fractions of the correct size are collected from the column 6 Place the column in a clean 15 ml conical tube containing a decapped 1 5 ml microcentrifuge tube 7 Apply the DNA sample to the column Centrifuge as before The purified probe is collected in the microcentrifuge tube B Selective precipitation of labelled DNA The following protocol leads to precipitation of DNA greater than about 20 nucleotides in length with unicorporated nucleotides remaining in solution Recovery of the labelled DNA by this method varies according to the DNA concentration and size and may be as low as 5096 1 Add one volume of 4 M ammonium acetate pH4 5 to the nick translation reaction and mix gently by pipetting up and down 2 Add four volumes of ethanol mix by inversion Chill the mixture for 15 minutes in a dry ice ethanol bath or place at 70 C for at least 30 minutes 3 Thaw the mixture if necessary by placing at 37 C for 2 minutes 4
18. ing reactions Check recovery of probe if purification is performed to remove unincorporated nucleotide 3 Low probe specific 3 If the specific activity activity 5 of the labelled DNA is lower than expected a labelling reaction should be carried out using a sample of the control DNA supplied with the system If this proceeds satisfactorily check the concentration and purity of your DNA Problem Possible cause 4 Loss of dNTP during evaporation 2 Non specific 1 Presence of background unincorporated over whole of label filter 36 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 reconstituted dNTP solution or by adsorption of the dNTP onto the walls of the tube If necessary the reconstituted dNTP solution may be counted and an adjustment made before setting up the labelling reaction 1 Unincorporated nucleotides can give high backgrounds Remove by Sephadex G 50 spin columns or ethanol precipitation see page 32 for protocol 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
19. lling systems are tested by our quality control group to ensure an incorporation rate greater than 55 after 10 minutes at 37 C The performance of RPN 1604 1605 is tested with the standard DNA provided using 17 pmol 25 ng DNA of a 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 o P dCTP 3000 Ci mmol code PB 10205 Incorporations greater than 55 are achieved after 10 minutes incubation at 37 C as assayed by thin ayer chromatography on PEI cellulose in 1 25 M KH PO PH3 4 n addition components of the kits are checked for identity by HPLC and the DNA solutions or concentration by UV spectrophotometry 3 System components Magaprime DNA RPN1604 RPN1605 RPN1606 RPN1607 labelling Primer solution 150 ul 300 ul 150 ul 300 ul Random nonamer primers in an aqueous solution Labelling buffer 300 ul 600 ul dATP dGTP and dTTP in Tris HCl pH7 5 2 mercaptoethanol and MgCl Nucleotide solutions a dATP 120 ul 240 ul b cCTP 120 ul 240 ul z c dGTP 120 ul 240 ul d dTTP 120 ul 240 ul gt in Tris HCl pH8 0 0 5 mM EDTA Reaction buffer 150 ul 300 ul A 10x concentrated buffer containing Tris HCl pH7 5 2 mercaptoethanol and MgCl Magaprime DNA RPN1604 labelling Enzyme solution 60 ul 1 unit ul DNA polymerase 1 Klenow fragment cloned in 100 mM potassium phosphate pH6 5
20. me of 50 ul 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 7 Avoid vigorous mixing of the for example 5ul a 32P dNTP reaction mixture as this can specific activity 3000 Ci mmol cause severe loss of enzyme Mix by gently pipetting up and activity down Spin for a few seconds in a microcentrifuge to bring the contents to the bottom of the tube 8 Incubate at 37 C for 8 Purified DNA can be labelled 10 minutes to high specific activity in 10 minutes at 37 C but if desired can be labelled for up to 1 hour at this temperature 17 Protocol Notes 8 Incubate at 37 C for 8 Continued 10 minutes 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 55S dNTP S are used 9 Stop the reaction by the 9 Labelled probe can be stored addition of 5 ul of 0 2 M at 15 C to 30 C in a non EDTA For use in a frost free freezer Prolonged hybridization denature the storage of P labelled labelled DNA by heating to probes can lead to substantial 95 100 C for 5 minutes then probe degradation 7 High chill on ice specific activity probes should
21. ons Figure 3 gives more complete information on their use in Megaprime reactions Reactions were carried out at 37 C for 5 minutes b Use of alternative specific activity o 5 P dNTPs When using a 32P dNTPs of specific activity 3000 Ci mmol the incubation time should be extended to 1 hour at 37 C 20 c Use of P dNTPoS When using 22S 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 Specific activity Figure 3a should be used to ascertain the number and quantity of labelled dNTP s required in order to prepare a probe of the desired specific activity 2 Efficiency Figure 3b indicates the efficiency of the chosen reaction conditions and thus permits a balance of specific activity and economy 3 Probe length Figure 3c gives a measure of mean probe lengths obtained under standard conditions Probe lengths were measured by denaturing agarose gel electrophoresis followed by autoradiography with reference to molecular weight standards Probe length can be affected by the concentration of DNA primer and nucleotide the size of the template DNA and also radiolysis of the labelled probe The data in t
22. ose blotting membranes Autoradiography products Hyperfilm high performance autoradiography films Hypercassettes and Hyperscreens available from stock Safety Products Radiation safety products for safe handling and storage of 3 P 33P 355 and 125 liquid scintillation products Agarose SepRate range of highly purified agarose for a range of DNA fragment sizes and users Labelled dNTPs See Table 2 39 Table 2 Labelled dNTPs and analogues available from GE Healthcare Compound Specific Activity Formulation Product TBq mmol Ci mmol see key code o 32P dATP 220 6000 1 PB 1074 110 3000 1 PB 10204 2 PB 204 30 800 PB 10384 15 400 1 PB 10164 2 PB 164 o 32PJ dCTP 220 6000 1 PB 10475 110 3000 2 PB 10205 1 PB 205 30 6000 1 PB 10385 15 400 2 PB 10165 PB 165 a 32P dGTP 220 3000 1 PB 10206 2 PB 206 30 800 2 PB 10386 15 400 1 PB 10166 2 PB 166 a 32P dTTP 110 300 1 PB 1027 2 PB 207 30 800 1 PB 10387 15 400 1 PB 10167 2 PB 167 a 32P dATP 37 110 1000 3000 1 BF 1001 55S dATPaS gt 37 gt 1000 1 SJ 1304 22 600 1 SJ 304 15 400 SJ 264 40 Compound Specific Activity Formulation Product TBq mmol Ci mmol see key code 55SJdCTPaS gt 37 gt 1000 1 J1305 22 600 1 SJ 305 15 400 1 SJ 265 35S dGTPaS 22 600 1 SJ 306 55S dTTPaS 22 600 1 SJ 307 8 3H dATP 0 37 1 1 10 30 2 TRK 347 1 2 2 8 5H dATP 1 83 3 7 50 100 2 TRK 633
23. rotocol 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 ul requiring a relatively high initial DNA concentration Protocol Notes 1 Fractionate restriction 1 A low melting point agarose endonuclease digested DNA of high purity for example in a suitable low melting SepRate LMP is point agarose gel containing recommended for maximum 0 5 ug ml ethidium bromide labelling efficiency 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 concentration 2 Excise the desired band 2 It is recommended that cleanly with the minimum the exposure to UV light of excess agarose and is minimized as prolonged transfer to a pre weighed exposure can damage the 1 5 ml microcentrifuge tube DNA 27 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 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 ul for the standard labelling protocol 5 Incubate the labelling reaction for 15 20 minutes at 37 C Notes 3 If the DNA is not to be used immedi
24. ss 2xSSC 30 mM Na handling In order to stabilize citrate 300 mM NaCl pH7 0 the paper the squares are using gentle agitation Rinse rinsed in ethanol briefly in distilled water and then once with ethanol for 5 minutes Then dry the filters under a heat lamp 29 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 DNA at the end of the reaction must be calculated Total amount of DNA A ng Total number of uCi added x 13 2 x 96 incorporation 25 Number of radioactive dNTPs added x average specific activity of dNTPs added This assumes a 2596 content of any one dNTP in the newly synthesized DNA and 25 ng of template DNA 13 2 equals four times the average molecular weight of the four dNTPs divided by 100 Notes 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 The mean value of the counts on the washed filter represents the proportion of the radionucleotide incorporated into the DNA probe while the mean of the unwashed filters represents the total amount of radioactivity in
25. st current information and a copy of the terms and conditions http www gehealthcare com lifesciences GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK 2 Handling 2 1 Safety warnings and precautions 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 Caution For use with radioactive material This product is to be used with radioactive material Please follow the manufacturer s instructions relating to the handling use storage and disposal of such material All chemicals should be considered as potentially hazardous We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice Wear suitable protective clothing such as laboratory overalls 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 2 2 Storage and stability Upon receipt of these systems components should be stored at 15 C to 30 C The components are stable for at least 3 months when stored under recommended conditions 2 3 Quality control The Megaprime DNA labe
26. uld not be less than 10 ul to maximize the efficiency of primer annedling The labelling efficiency of a DNA sample can be compared with that of the standard DNA supplied with the kit In this case 5 ul of standard DNA should be used Protocol 3 Place 25 ng 5 ul of template DNA into a clean microcentrifuge tube and to it add 5 ul of primers Denature by heating to 95 100 C for 5 minutes in a boiling water bath Spin briefly in a microcentrifuge to bring the contents to the bottom of the tube Keeping the tube at room temperature add the nucleotides and 10x reaction buffer RPN 1604 5 or the labelling buffer RPN 1606 7 water and enzyme Notes If the volume of DNA and primers is less than 10 ul 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 The volume of low melting point agarose DNA should not exceed 25 yl in a 50 ul reaction The enzyme can be added directly to the reaction mix or pipetted on to the side of the microcentrifuge tube and washed down with the water Component RPN1604 5 RPN1606 7 Labelling 10 ul buffer Unlabelled 4 ul of each dNTPs omitting those to be used as label 16 Protocol Notes Reaction Sul buffer Enzyme 2 ul 2 ul Water as appropriate for a final reaction volu
27. used in combination 22 a Specific activity g 5 iii E E o 4 ii o ocd me 5 i oD 2 a wl x o oW 51 9 ae na O 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 32P dNTPs in the Megaprime DNA labelling system see notes on page 26 23 b Incorporation efficiency 100 oO 3 80 QD uo 60 o e amp 40 i B ii A 9 20 ii w a 0 10 20 30 40 50 60 70 80 90 100 Total input label pmols One labelled dNTP ii Two labelled dNTP iii Three labelled dNTP Figure 3 The use of a 32P dNTPs in the Megaprime DNA labelling system see notes on page 26 24 c Probe length _ 100 wn G 3 80 5 m d 2 40 di o iii o S 20 d 0 10 20 30 40 50 60 70 80 90 100 Total input label pmols One labelled dNTP ii Two labelled dNTP iii Three labelled dNTP Figure 3 The use of a 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 may deviate 10 from those shown b As the number of different labelled nucleotides is increased at a given level of total input label the net synthesis of DNA is reduced Although the overall incorporation eff
28. volume may be scaled up or down if more or less than 25 ng of DNA is to be labelled Protocol Component RPN1604 5 RPN1606 7 Labelling 10 ul buffer Unlabelled 4plofeach dNTPs omitting those to be used as label Reaction Sul buffer Radiolabelled dNTP 5 ul 5 ul dCTP Enzyme 2 ul 2yl 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 7 Incubate at 37 C for 10 minutes 13 Notes Avoid vigorous mixing of the reaction mixture as this can cause severe loss of enzyme activity Purified DNA can be labelled to high specific activity in 10 minutes at 37 C but if desired can be labelled for up to 1 hour at this temperature When labelling DNA in low melting point agarose longer incubation of 15 30 minutes at 37 C are required for optimum labelling Longer incubation Protocol 7 Incubate at 37 C for 10 minutes continued 8 Stop the reaction by the addition of 5 ul 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 14 Notes 7 Continued times up to 60 minutes are required when nucleotide analogues e g 55S 4NTPaS are used Labelled probe can be stored at 15 C to 30 C in a non frost free freezer Prolonged storage of P labelled probes can lead to substantial probe degradation 7
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