GE RPN1607 - Manual

Magaprime DNA RPN1604 RPN1605 RPN1606 RPN1607 labelling Enzyme solution; 60 µl 120 µl 60 µl 120 µl 1 unit/µl DNApolymerase 1 Klenowfragment (cloned in100 mM potassiumphosphate pH6.5, 10 mM 2-mercapto-ethanol and50% glycerol Standard DNA 25 µl 50 µl 25 µl 50 µl solution; 5 ng/µl Hin d III digested la...

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 primer-template complex is a substrate for the ‘Klenow’ fragment of DNA polymerase 1. By substituting a radiola...

1 0 Figur e 1. Pr eparation of labelled pr obes using GE Healthcar e’s megaprime DNA labelling systems. Linear dsDNA Denatur e in pr esence of monamer primers Add Multiprime DNA reaction buffer Add labelled dNTP and ‘Klenow ’ DNA polymerase. Incubate Denatur e to r elease labelled pr obe and add dir...

1 1 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 EDTA (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 ca...

1 5 Protocol 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. Notes 8. Continued under the conditions given above is not required with the isotopes 32 P and 33 P. Purif...

1 9 Protocol 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 . Notes 9. Continued described in Appendix III. Calculation of probe specific activity is described in Appe...

5.3. Use of alternative reaction conditions a. Use of more than one labelled [ α – 32 P]dNTP. Table 1 lists the results 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. Reacti...

b) Incorporation efficiency i) One labelled dNTPii) Two labelled dNTPiii) Three labelled dNTP Figure 3. The use of [ α – 32 P]dNTPs in the Megaprime DNA labelling system (see notes on page 26). 2 4 P e rcentage of added label 100 80 60 40 20 (i)(ii)(iii) 0 10 20 30 40 50 60 70 80 90 100 Total input ...

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 ...

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 5 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...

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]dTTP α S ~22 ~600 1 SJ 307 [8– 3 H]dATP 0.37–1.1 10–30 2 TRK 347 [1’,2’,2,8– 3 H]dATP 1.83–3.7 50–100 2...

imagination at work RPN1604PL Rev B 2006 http://www.gehealthcare.com/lifesciences GE Healthcare UK LimitedAmersham Place, Little Chalfont , Buckinghamshire, HP7 9NAUK GE Healthcare regional office contact numbers: Asia Pacific Tel: +85 65 62751830 Fax: +85 65 62751829 Australasia Tel: + 61 2 8820 82...