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. 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 [α–32P]dNTPs.
When using [α–32P]dNTPs of specific activity <3000 Ci/mmol the
incubation time should be extended to 1 hour at 37°C.
20
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.
% 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