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YOSHIO KANEMITSU AND TAKEHlKO KOJl
10. Incubate in moist chamber overnight at 40°C.
11. Wash in 50% formamide in 2x SSC Ihr 40°C,S times.
12. Wash in PBS 10 mins RT, twice.
13. Put the blocking solution (0.5 mg/ml normal mouse IgG and
5% BSA in PBS) on the section and incubate 1 hr RT in moist
chamber.
14. Remove the blocking solution and put the anti-TT solution
(HRP conjugated mouse anti-(T-T dimer) antibody in
blocking solution) on the section. Incubate 2 hrs RT in moist
chamber.
15. Wash in PBS 10 mins RT 5 times.
16. Visualize the signals in the DAB solution (DAB, nickel and
cobalt in phosphate buffer).
Results
Evaluation of the PCR product
Typical results of the analysis of asymmetric PCR product are
shown in Figure 2. It shows that two-step asymmetric PCR generates five times more ssDNA (second peak) than dsDNA (first
peak) in weight. (It equals ten times in molarity.) Single-step
asymmetric PCR produces smaller amounts of ssDNA and
the ssDNA/dsDNA ratio is lower.
Gel electrophoresis of the second PCR product is shown in
Figure 3. ssDNA runs relatively slower than ds DNA and its
band is not sharp because of its complex second structure.
The intensity of the band of ss DNA is relatively obscure
when it is stained with ethidium bromide which visualizes the
double stranded part. The quantity of ss DNA product increases
not exponentially but linearly, and it often increases over 50 cycles.