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Add 40 µl diluted hybridized DNA (4 µg) to each tube. Place tubes in thermal cycler
set at 72°C.
This reforms priming sites at both ends of tester:tester complexes necessary for exponential
amplification of difference products.
37. Add 3 µl Taq DNA polymerase, mix by pipetting, and incubate an additional 5 min.
38. Add 10 µl 24-mer primer (O primer set), mix by pipetting, and overlay with mineral
oil.
If using a 96-well thermal cycler, double the number of tubes and halve the PCR recipe in
each tube. In this case, addition of mineral oil is not necessary.
39. Perform the following two-step PCR program:
10 cycles:
Final step:
1 min
3 min
10 min
95°C
72°C
72°C
(denaturation)
(extension)
(extension).
For the OBgl 24 primer, a lower annealing temperature of 70°C is required.
40. Remove as much mineral oil as possible and combine the contents of the PCR tubes
in a microcentrifuge tube. Extract and isopropanol precipitate as described (step 13),
but dissolve the pellet in 40 µl water and do not pool DNA.
41. Digest single-stranded templates with mung bean nuclease (MBN) by mixing:
14 µl water
4 µl 10× mung bean nuclease buffer
20 µl amplified difference product
2 µl 10 U/µl mung bean nuclease (MBN)
40 µl total volume.
Incubate at 30°C for 30 min.
42. Add 160 µl of 50 mM Tris⋅Cl, pH 8.9. Inactivate MBN by incubating 5 min at 98°C.
43. Prepare two tubes of PCR mix (360 µl) containing the O 24-mer primer as in step 8.
Add 40 µl MBN-treated difference product in each tube and place in a thermal cycler
set at 72°C.
For OBgl 24-mer use an annealing temperature of 70°C.
44. Add 3 µl of 5 U/µl (15 U) Taq DNA polymerase to each tube, mix by pipetting,
overlay with 110 µl mineral oil, and incubate 5 min at 72°C.
Again, double the number of PCR tubes and halve the given recipe placed in each tube if
using a 96-well PCR machine.
45. Perform the following two-step PCR program:
20 cycles:
Final step:
1 min
3 min
10 min
95°C
72°C
72°C
(denaturation)
(extension)
(extension).
For the OBgl 24 primer, a lower annealing temperature of 70°C is required.
46. Run 10 µl amplified product on a 2% agarose gel with DNA concentration standards
(UNIT 2.5A). If necessary to improve the yield, perform 1 to 3 more cycles after addition
of 3 µl fresh Taq DNA polymerase.
Discovery of
Differentially
Expressed Genes
25B.7.7
Current Protocols in Molecular Biology
Supplement 60