GE RPN1605 Manual do Utilizador

GE HealthcareAmershamMegaprime™ DNA Labelling SystemsProduct BookletCodes: RPN1604 RPN1605 RPN1606 RPN1607

10Figure 1. Preparation of labelled probes using GE Healthcare’s megaprime DNA labelling systems.Linear dsDNADenature in presenceof monamer primersAdd

11Protocol1. 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

12Protocol2. Place the required tubes from the Megaprime system, with the exception of the enzyme, at room temperature to thaw. Leave th

13ProtocolComponent RPN1604/5 RPN1606/7Labelling 10 µlbuffer Unlabelled 4 µl of each –dNTPs omitting those to be used as labelReaction 5 µl

14Protocol7. Incubate at 37°C for 10 minutes continued.8. Stop the reaction by the addition of 5 µl of 0.2 M EDTA. For use in a hybridizat

15Protocol8. 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

16Protocol3. Place 25 ng (5 µl) of template DNA into a clean microcentrifuge tube and to it add 5 µl of primers. Denature by heatin

17Protocol Reaction 5 µl –buffer Enzyme 2 µl 2 µlWater* as appropriate for a final reaction volume of 50 µl** When calculating this vol

18Protocol8. Incubate at 37°C for 10 minutes continued.9. Stop the reaction by the addition of 5 µl of 0.2 M EDTA. For use in a hybridi

19Protocol9. Stop the reaction by the addition of 5 µl of 0.2 M EDTA. For use in a hybridization, denature the labelled DNA by heat

2Page finder1. Legal 32. Handling 4 2.1. Safety warnings and precautions 4 2.2. Storage and stability 4 2.3. Quality control 43. System comp

5.3. Use of alternative reaction conditionsa. Use of more than one labelled [α–32P]dNTP.Table 1 lists the results of a selection of standard reactions

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 temperatu

22a. 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

a) Specific activityi) One labelled dNTPii) Two labelled dNTPiii) Three labelled dNTPFigure 3. The use of [α–32P]dNTPs in the Megaprime DNA labell

b) Incorporation efficiencyi) One labelled dNTPii) Two labelled dNTPiii) Three labelled dNTPFigure 3. The use of [α–32P]dNTPs in the Megaprime DNA

25c) Probe lengthi) One labelled dNTPii) Two labelled dNTPiii) Three labelled dNTPFigure 3. The use of [α–32P]dNTPs in the Megaprime DNA labelling

c. The data was generated using the standard labelling protocols. If dNTPs <3000 Ci/mmol are to be used, then the desired probe specific acti

27Protocol1. Fractionate restriction endonuclease digested DNA in a suitable low melting point agarose gel containing 0.5 µg/ml ethidi

28Protocol3. 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 denatur

29Protocol1. 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/

1. LegalGE and GE monogram are trademarks of General Electric Company. Amersham, Megaprime, Hybond, Hyperfilm, Hypercassette, Hyperscreen, Sensitize,

30Protocol 5. Place the squares in separate vials with at least 5 ml of scintillation fluid and count.6. Efficiency of counting will vary,

31Protocol 6. Continued.The amount of radioactivity incorporated during the reaction (B) in dpm.B = total number of µCi added x 2.2x104 x % incorporat

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

any liquid from the microcentrifuge tube. Refill with Sephadex and centrifuge as before. Continue until the column is packed to a volume of 1

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

35Problem1. Low signalPossible cause1. Incomplete denaturation of template DNA2. Low probe concentration 3. Low probe specific activityRem

36Problem2. Non-specific background over whole of filterPossible cause4. Loss of dNTP during evaporation1. Presence of unincorporated

37Problem Possible cause2. Concentrated probe has contacted membrane directly during probe addition3. Probe concentration is too high4.

8. References1. FEINBERG, A.P. and VOGELSTEIN, B., Anal. Biochem., 132, pp.6-13, 1983.2. FEINBERG, A.P. and VOGELSTEIN, B., Addendum Anal. Biochem

9. Related ProductsLabelling systemsNick translation kits N5000/55003’-end labelling kit N40205’ end labelling kit RPN 1509RNA labelling system(pai

42. Handling 2.1. Safety warnings and precautionsWarning: For research use only. Not recommended or intended for diagnosis of disease in humans or ani

Table 2. Labelled dNTPs and analogues available from GE HealthcareCompound Specific Activity Formulation P

Compound Specific Activity Formulation Product TBq/mmol Ci/mmol (see key) code[35S]dCTPαS >37 &g

imagination at workRPN1604PL Rev B 2006http://www.gehealthcare.com/lifesciencesGE Healthcare UK LimitedAmersham Place, Little Chalfont, Buckinghams

5using 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

3. System componentsMagaprime DNA RPN1604 RPN1605 RPN1606 RPN1607labellingPrimer solution: 150 µl 300 µl 150 µl 300 µlRandom nonamerprimers i

Magaprime DNA RPN1604 RPN1605 RPN1606 RPN1607labellingEnzyme solution; 60 µl 120 µl 60 µl 120 µl1 unit/µl DNApolymerase 1 Klenowfragment (clo

3.1. Megaprime DNA labelling systems30 standard labelling reactions – for use with any radioactive nucleotide RPN 160460 standard labelling react

4. IntroductionFeinbereg and Vogelstein (1,2) introduced the use of random sequence hexancleotides to prime DNA synthesis on denatured template DNA at