John M. S. Bartlett.pdf - Bio-Nica.info

370 Shen et al.
3. Mineral oil.
4. Gel-purification kits/reagents, such as QIAEX Gel Extraction Kit (Qiagen #20020,
Chatsworth, CA) or QiaOuick Gel Extraction Kit (Qiagen #28704).
5. All buffers and solutions must be free of DNase.
6. α- 35 S-dATP (10 µCi/µL 1000 Ci/mmol; Amersham Corp).
7. Sequencing primers (degenerate primers) dissolved in H 2 O, or 0.1× TE buffer.
The sequencing reaction reagents can be homemade. However, we recommend purchasing
them from a commercial company to ensure uniform performance. In the following
materials, we include the catalog number for US Biochemicals (Cleveland, OH).
8. Reaction buffer (USB #71030): 260 mM Tris-HCl, pH 9.5; 65 mM MgCl 2 .
9. ∆Taq DNA polymerase (USB #71059) or Taq DNA polymerase, (USB# 71057): 32 U/µL.
10. Taq DNA polymerase dilution buffer (USB #71051): 10 mM Tris-HCl, pH 8.0, 1 mM
2-mercaptoethanol, 0.5% Tween-20, and 0.5% Nonidet P-40.
11. Four separate primer label mixes: dGTP label mix: 3.0 µM (USB #71034); dATP label
mix: 3.0 µM (USB #71036); dTTP label mix: 3.0 µM (USB #71037); and dCTP label
mix: 3.0 µM (USB #71038).
12. Four separate termination mixes: ddG terminator mix: 15 µM each dGTP, dATP, dTTP,
dCTP, and 22.5 µM ddGTP (USB #71020); ddA termination mix: 15 µM each dGTP, dATP,
dTTP, dCTP, and 300 µM ddATP (USB #71035); ddT termination mix: 15 µM each dGTP,
dATP, dTTP, dCTP, and 450 µM ddTTP (USB #71040); and ddC terminator mix: 15 µM
each dGTP, dATP, dTTP, dCTP, and 75 µM ddCTP (USB #71025).
13. Stop/gel-loading solution (USB #70724): 95% formamide, 20 mM EDTA, 0.05% bromophenol
blue, and 0.05% xylene cyanol FF.
14. 1× TE buffer: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.
3. Methods
3.1. Preparation of DNA as a Sequencing Template (see Note 2)
1. After gel electrophoresis, PCR fragment(s) of interest is cut out from the gel.
2. DNA in the gel is purified with the Qiagen gel-purification kit, and final PCR products are
resuspended in a proper amount of 0.1× TE buffer.
3. To estimate the amount of PCR product, run an aliquot of the PCR products on an agarose
gel. The amount of DNA may be estimated by a comparison with the amount of DNA
that was used in the mol-wt ladder.
In the following steps, always keep tubes on ice, unless otherwise indicated.
4. Prepare the following labeling PCR mix (see Note 3):
H 2 O 10–8 µL
DNA (in 0.1× TE) (need total of 25–100 ng) 11–9 µL
Reaction buffer 12 µL
Degenerate primers (5–200 µM) 11 µL
dGTP label mix 11 µL
dCTP label mix 11 µL
dTTP label mix 11 µL
α- 35 S-dATP (10 µCi/µL >1000 Ci/mmol) 10.5 µL
Taq DNA polymerase (4 U/µL) (diluted in Taq dilution buffer) 12 µL
Total volume 17.5 µL
Cover the label PCR mix with 10–20 µL of mineral oil.
3.2. Labeling PCR (see Note 4)
1. Run the following PCR program: presoak at 94°C for 3 to 5 min followed by 45 cycles
of 95°C for 30 s and 52°C for 30 s.