GE Label Maker RPN1605 User Manual

GE Healthcare  
Amersham  
Megaprime™ DNA  
Labelling Systems  
Product Booklet  
Codes:  
RPN1604  
RPN1605  
RPN1606  
RPN1607  
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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 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 most current information  
and a copy of the terms and conditions  
GE Healthcare UK Limited.  
Amersham Place, Little Chalfont,  
Buckinghamshire, HP7 9NA UK  
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2. Handling  
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.  
2.1. 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.  
2.2. Storage and  
stability  
Caution: For use with  
radioactive material.  
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.  
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,  
2.3. Quality control  
The Megaprime DNA labelling  
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  
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using 17 pmol/25 ng DNA of  
[α32P] labelled nucleotides,  
specific activity 3000 Ci/mmol  
(codes PB 10204-7) and  
RPN 1606/1607 are tested  
using 17 pmol/25 ng DNA of  
[α32P]dCTP, 3000 Ci/mmol  
(code PB 10205). Incorporations  
greater than 55% are achieved  
after 10 minutes incubation  
at 37°C, as assayed by thin-  
layer 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.  
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3. System components  
Magaprime DNA  
RPN1604 RPN1605 RPN1606 RPN1607  
labelling  
Primer solution:  
Random nonamer  
primers in an  
150 µl  
300 µl  
150 µl  
300 µl  
aqueous solution  
Labelling buffer;  
dATP, dGTP and  
dTTP in Tris/HCl  
pH7.5,  
300 µl  
600 µl  
2-mercaptoethanol  
and MgCl2  
Nucleotide solutions  
(a) dATP  
(b) cCTP  
(c) dGTP  
(d) dTTP  
120 µl  
120 µl  
120 µl  
120 µl  
240 µl  
240 µl  
240 µl  
240 µl  
in Tris/HCl pH8.0,  
0.5 mM EDTA  
Reaction buffer:  
A 10x concentrated  
buffer containing  
Tris/HCl pH7.5,  
150 µl  
300 µl  
2-mercaptoethanol  
and MgCl2  
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Magaprime DNA  
labelling  
RPN1604 RPN1605 RPN1606 RPN1607  
Enzyme solution;  
1 unit/µl DNA  
60 µl  
120 µl  
60 µl  
120 µl  
polymerase 1 Klenow  
fragment (cloned in  
100 mM potassium  
phosphate pH6.5,  
10 mM 2-mercapto-  
ethanol and  
50% glycerol  
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  
1.25 ml  
2.5 ml  
1.25 ml  
2.5 ml  
solution; 500ng/ml  
sonicated herring  
sperm DNA in  
10 mM Tris/HCl  
pH 8.0, 1 mM  
EDTA  
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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  
RPN 1606  
RPN 1607  
30 standard labelling reactions –  
for use with radioactively labelled dCTP  
60 standard labelling reactions –  
for use with radioactively labelled dCTP  
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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 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  
that 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 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  
Magaprime 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 1.9x109 dpm/µg can be produced with  
the majority of DNA substrates, using the standard protocol, after  
10 minutes incubation at 37°C. This rapid labelling is achieved by  
the use of nonamer primers rather than the conventional hexamers  
(Figure 1). Nonamers allow for more efficient priming from the  
template 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  
low melting point agarose takes only 15–30 minutes in contrast to  
conventional systems where overnight incubation are necessary.  
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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 pmol[α–32P]dNTP,  
specific activity 3000 Ci/mmol. For alternative reaction conditions  
refer to page 20.  
DNA prepared by standard minilysate 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/HCl,  
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 Megaprime protocol  
Protocol  
Notes  
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).  
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.  
11  
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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  
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 50 µl in the final  
Megaprime reaction.  
Denature by heating to  
95–100°C for 5 minutes  
in a boiling water bath.  
volume of low melting point  
agarose DNA should not  
exceed 25 µl in a 50 µl reaction.  
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:  
5. The reaction volume may be  
scaled up or down if more or  
less than 25 ng of DNA is to  
be labelled.  
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Protocol  
Notes  
Component  
Labelling  
buffer  
RPN1604/5 RPN1606/7  
10 µl  
Unlabelled  
dNTPs  
4 µl of each  
omitting  
those to be  
used as  
label  
Reaction  
buffer  
5 µl  
Radiolabelled  
(dNTP)  
5 µl  
2 µl  
5 µl (dCTP)  
2 µl  
Enzyme  
6. Avoid vigorous mixing of the  
reaction mixture as this can  
cause severe loss of enzyme  
activity.  
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  
7. 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  
13  
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Protocol  
Notes  
7. Continued.  
7. Incubate at 37°C for  
10 minutes 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). High  
specific activity probes  
should be stored for no  
longer than 3 days. Although  
probe purification is not  
usually necessary for most  
membrane applications,  
the removal of unicorporated  
nucleotide is sometimes  
useful to reduce background  
in filter hybridizations  
for probes >109 dpm/µg or  
when the reaction yields an  
incorporation of less than 50%.  
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  
14  
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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. Dilute the DNA to a  
concentration of 5 ng/µl in  
either distilled water or  
10 mM TE buffer.  
1. DNA solutions at  
concentrations in the range  
5–25 ng/µl can be used if desired.  
However the denaturing  
volume (step 3) should not be  
less than 10 µl to maximize  
the efficiency of primer  
annealing. 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.  
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.  
15  
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Protocol  
Notes  
3. Place 25 ng (5 µl) of  
template DNA into a clean  
microcentrifuge tube and to  
it add 5 µl of primers.  
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  
Denature by heating to  
95–100°C for 5 minutes in a  
boiling water bath.  
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 µl in a 50 µl  
reaction.  
4. Spin briefly in a  
microcentrifuge to bring the  
contents to the bottom of  
the tube.  
5. Keeping the tube at  
5. 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.  
room temperature add the  
nucleotides and 10x  
reaction buffer (RPN 1604/5)  
or the labelling buffer (RPN  
1606/7), water and enzyme:-  
Component  
Labelling  
buffer  
RPN1604/5 RPN1606/7  
10 µl  
Unlabelled  
dNTPs  
4 µl of each –  
omitting  
those to be  
used as label  
16  
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Protocol  
Notes  
Reaction  
buffer  
5 µl  
2 µl  
Enzyme  
Water*  
2 µl  
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 [α32P]dNTP,  
specific activity 3000 Ci/mmol.  
Mix by gently pipetting up and  
down. Spin for a few seconds  
in a microcentrifuge to bring  
the contents to the bottom of  
the tube.  
7. Avoid vigorous mixing of the  
reaction mixture as this can  
cause severe loss of enzyme  
activity.  
8. 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  
8. Incubate at 37°C for  
10 minutes.  
temperature.  
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Protocol  
Notes  
8. Continued  
8. Incubate at 37°C for  
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. [35S]dNTP(S)  
are used.  
9. 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). High  
specific activity probes  
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.  
should 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 >109 dpm/µg or  
when the reaction yields an  
incorporation of less  
than 50%. This procedure is  
18  
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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.  
Purification of 32S labelled  
probes is however required  
to reduce filter background.  
19  
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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 [α32P]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 [α32P]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.  
Reactions were carried out at 37°C for 5 minutes.  
b. Use of alternative specific activity [α32P]dNTPs.  
When using [α32P]dNTPs of specific activity <3000 Ci/mmol the  
incubation time should be extended to 1 hour at 37°C.  
20  
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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. 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 the 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.  
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a) Specific activity  
(iii)  
(ii)  
(i)  
5
4
3
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 [α32P]dNTPs in the Megaprime DNA labelling  
system (see notes on page 26).  
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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 [α32P]dNTPs in the Megaprime DNA labelling  
system (see notes on page 26).  
24  
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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 [α32P]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 efficiency is reduced the  
labelled product is of a higher specific activity.  
25  
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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  
specific activity of  
dNTP to be used  
x
required probe  
specific activity  
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 µl =  
pmol of dNTP required x specific activity of dNTP (Ci/mmol) x 10–3  
radioactive concentration of dNTP (mCi/ml)  
26  
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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.  
Notes  
Protocol  
1. A low melting point agarose  
of high purity for example  
SepRate-LMP is  
recommended for maximum  
labelling efficiency.  
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 concentration  
2. Excise the desired band  
cleanly, with the minimum  
of excess agarose and  
transfer to a pre-weighed  
1.5 ml microcentrifuge tube.  
2. It is recommended that  
the exposure to UV light  
is minimized, as prolonged  
exposure can damage the  
DNA.  
27  
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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). The volume of  
DNA should not exceed 25 µl  
for the standard labelling  
protocol.  
4. 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.  
5. Incubate the labelling  
reaction for 15–20 minutes  
at 37°C.  
6.2. Appendix II. 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  
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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 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. In aqueous solution DE81  
paper becomes fragile and  
care should be taken when  
handling. In order to stabilize  
the paper the squares are  
rinsed in ethanol.  
4. Wash the remaining two  
filters twice for 5 minutes  
each, at room temperature  
in excess 2xSSC (30 mM Na3  
citrate, 300 mM NaCl pH7.0)  
using gentle agitation. Rinse  
briefly in distilled water and  
then once with ethanol for  
5 minutes. Then dry the  
filters under a heat lamp.  
29  
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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. 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.  
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 the reaction  
mix, such that;  
% incorporation = mean counts on washed filters x 100  
mean counts on unwashed filters  
Total amount of DNA (A) ng =  
Total number of µCi added x 13.2* x % incorporation + 25  
Number of radioactive dNTPs added x average specific  
activity of dNTPs added  
This assumes a 25% 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.  
30  
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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. Transfer 1–10 µl of diluted reaction mixture to two duplicate tubes  
containing 200 µl water or 0.2M EDTA and 50 µl 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.  
5. Collect the precipitated DNA by vacuum filtration on a glass fibre  
or nitrocellulose filter disc.  
31  
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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.  
32  
7. Count the dried filter discs by liquid scintillation or Cerenkov ( P)  
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. 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  
32  
33  
incorporation is less than 50%. If P or P-labelled probes are to  
be used in combination with GE Healthcare’s new Rapid-hyb 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, in which a decapped 1.5 ml microcentrifuge tube  
has been inserted. Centrifuge at 1600 g for 5 minutes. Remove  
32  
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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. 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 50%.  
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. 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, 67% ethanol at room temperature by gentle inversion,  
centrifugation and aspiration.  
33  
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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)  
0.2 M EDTA solution  
Adjustable pipettes for example Pipetman™  
Sterile pipette tips  
Waterbaths at 37°C and 100°C  
Polypropylene microcentrifuge tubes  
Microcentrifuge  
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 Na3 citrate, 300 mM NaCl, pH 7.0)  
™ Pipetman is a registered trademark of Gilson  
34  
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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. If the specific activity  
activity  
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.  
35  
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Problem  
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. If  
necessary the  
reconstituted dNTP  
solution may be  
counted and an  
adjustment made  
before setting up the  
labelling reaction.  
2. Non-specific  
background  
over whole of  
filter  
1. Presence of  
unincorporated  
label  
1. Unincorporated  
nucleotides can give  
high backgrounds.  
Remove by Sephadex  
G-50 spin columns  
or ethanol  
precipitation (see  
page 32 for protocol)  
36  
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Problem  
Possible cause  
Remedy  
2. Concentrated  
probe has contacted  
membrane  
directly during  
probe addition  
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  
container in an 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  
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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., 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; A Practical Approach Vol 2 (T.A. Brown, ed)  
Oxford University Press, Oxford, 1991. pp.57-109.  
38  
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9. Related Products  
Labelling systems  
Nick translation kits  
3’-end labelling kit  
N5000/5500  
N4020  
5’ end labelling kit  
RPN 1509  
RNA labelling system  
(paired promoter SP6/T7 system)  
RPN 3100  
Hybridization buffers  
Rapid-hyb buffer  
Hybridization buffer tablets  
RPN 1635/6  
RPN 131  
Hybridization membranes  
Hybond™ - Range of nylon and nitrocellulose  
blotting membranes  
Autoradiography products  
Hyperfilm™ - high performance autoradiography films  
Hypercassettes™ and Hyperscreens™ - available from stock  
Safety Products  
32 33  
Radiation safety products for safe handling and storage of P/ P/  
35  
125  
S and  
I, 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  
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Table 2. Labelled dNTPs and analogues available from GE Healthcare  
Compound  
Specific Activity  
TBq/mmol  
Formulation  
(see key)  
Product  
code  
Ci/mmol  
32  
[α– P]dATP  
~220  
~110  
~6000  
~3000  
1
1
2
1
1
2
PB 1074  
PB 10204  
PB 204  
PB 10384  
PB 10164  
PB 164  
~30  
~15  
~800  
~400  
32  
[α– P]dCTP  
~220  
~110  
~6000  
~3000  
1
2
1
1
2
PB 10475  
PB 10205  
PB 205  
PB 10385  
PB 10165  
PB 165  
~30  
~15  
~6000  
~400  
32  
[α– P]dGTP  
~220  
~3000  
1
2
1
1
2
PB 10206  
PB 206  
PB 10386  
PB 10166  
PB 166  
~30  
~15  
~800  
~400  
32  
[α– P]dTTP  
~110  
~300  
1
2
1
1
2
PB 1027  
PB 207  
PB 10387  
PB 10167  
PB 167  
~30  
~15  
~800  
~400  
32  
[α– P] dATP  
37–110 1000–3000  
1
BF 1001  
35  
[
S]dATPαS  
>37  
~22  
~15  
>1000  
~600  
~400  
1
1
1
SJ 1304  
SJ 304  
SJ 264  
40  
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Compound  
Specific Activity  
TBq/mmol  
Formulation  
(see key)  
Product  
code  
Ci/mmol  
35  
[
S]dCTPαS  
>37  
~22  
~15  
>1000  
~600  
~400  
1
1
1
SJ1305  
SJ 305  
SJ 265  
35  
35  
[
[
S]dGTPαS  
~22  
~22  
~600  
~600  
1
1
SJ 306  
SJ 307  
S]dTTPαS  
3
[8– H]dATP  
0.37–1.1  
1.83–3.7 50–100  
1.85–3.14 50–85  
0.55–1.1  
0.185–0.740 5–20  
0.9–1.85 25–50  
10–30  
2
2
2
2
2
2
2
2
2
3
TRK 347  
TRK 633  
TRK 625  
TRK 352  
TRK 350  
TRK 627  
TRK 576  
TRK 424  
TRK 354  
IM 5103  
3
[1’,2’,2,8– H]dATP  
3
[1’,2’,5– H]dCTP  
3
[5– H]dCTP  
15–30  
3
[8– H]dGTP  
3
[1’2,(– H]dGTP  
[methyl, 1’,2’– H]TTP  
[methyl– H]TTP  
3
3.3–4.8 90–130  
3
40–60  
30  
>1500  
125  
[
I]dCTP  
>55  
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  
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France  
Portugal  
GE Healthcare offices:  
GE Healthcare  
regional office  
contact numbers:  
Tel: 01 69 35 67 00  
Fax: 01 69 41 98 77  
Tel: 21 417 7035  
Fax: 21 417 3184  
GE Healthcare Bio-Sciences AB  
Björkgatan 30 751 84  
Uppsala  
Germany  
Russia, C.I.S. & N.I.S  
Tel: +7 495 956 5177  
Fax: +7 495 956 5176  
Asia Pacific  
Tel: 0800 9080 711  
Fax: 0800 9080 712  
Sweden  
Tel: +85 65 62751830  
Fax: +85 65 62751829  
GE Healthcare Europe GmbH  
Munzinger Strasse 5 D-79111  
Freiburg  
Greater China  
Spain  
Australasia  
Tel: +852 2100 6300  
Fax: +852 2100 6338  
Tel: 902 11 72 65  
Fax: 935 94 49 65  
Tel: + 61 2 8820 8299  
Fax: +61 2 8820 8200  
Germany  
Italy  
Sweden  
GE Healthcare UK Limited  
Amersham Place  
Little Chalfont  
Buckinghamshire  
HP7 9NA  
Austria  
Tel: 02 26001 320  
Fax: 02 26001 399  
Tel: 018 612 1900  
Fax: 018 612 1910  
Tel: 01/57606-1613  
Fax: 01/57606-1614  
Japan  
Switzerland  
Belgium  
Tel: +81 3 5331 9336  
Fax: +81 3 5331 9370  
Tel: 0848 8028 10  
Fax: 0848 8028 11  
Tel: 0800 73 890  
Fax: 02 416 8206  
UK  
GE Healthcare Bio-Sciences  
Corp  
Korea  
UK  
Canada  
Tel: 82 2 6201 3700  
Fax: 82 2 6201 3803  
Tel: 0800 515 313  
Fax: 0800 616 927  
Tel: 1 800 463 5800  
Fax: 1 800 567 1008  
800 Centennial Avenue  
P.O. Box 1327  
Piscataway  
Latin America  
USA  
Central, East, & South  
East Europe  
Tel: +55 11 3933 7300  
Fax: + 55 11 3933 7304  
Tel: +1 800 526 3593  
Fax: +1 877 295 8102  
NJ 08855-1327  
USA  
Tel: +43 1 972 720  
Fax: +43 1 972 722 750  
Middle East & Africa  
Tel: +30 210 96 00 687  
Fax: +30 210 96 00 693  
GE Healthcare Bio-Sciences KK  
Sanken Bldg. 3-25-1  
Hyakunincho Shinjuku-ku  
Tokyo 169-0073  
Denmark  
Tel: 45 70 25 24 50  
Fax: 45 45 16 2424  
Netherlands  
Tel: 0800-82 82 82 1  
Fax: 0800-82 82 82 4  
Eire  
Japan  
Tel: 1 800 709992  
Fax: +44 1494 542010  
Norway  
Tel: +47 815 65 777  
Fax: +47 815 65 666  
Finland & Baltics  
Tel: +358 9 512 3940  
Fax: +358 9 512 39439  
GE Healthcare UK Limited  
Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA  
UK  
imagination at work  
RPN1604PL Rev B 2006  
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