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

316 Aubele and Smida
2.2. Chemicals Required for DOP-PCR
1. DOP-primer: 5′- CCG ACT CGA GNN NNN NAT GTG G - 3′, with N = A, C, G, or T in
approx equal proportions, (Biometra, Göttingen, Germany).
2. Topoisomerase I (Life Technologies, Eggenstein, Germany).
3. Taq DNA Polymerase (Perkin–Elmer Life Sciences, Maryland).
4. DNTPs (10 mM each of dATP, dCTP, dGTP, and TTP; Sigma-Aldrich, Steinheim,
Germany).
5. 10× amplification buffer: 500 mM KCl, 200 mM Tris-HCl, pH 8.4.
6. 50 mM MgCl 2 .
3. Method
3.1. Preparation of Tumor Samples
1. Dewax 5- to 10-µm thick paraffin sections for 30 min in a Coplin jar with Xylene.
2. Transfer slides to a coplin jar with fresh xylene for 5 min.
3. Transfer slides to a Coplin jar with 100% EtOH for 5 min.
4. Transfer slides to a coplin jar with 70% EtOH for 5 min.
5. Transfer slides to a coplin jar with 50% EtOH for 5 min.
6. Transfer slides to a coplin jar with distilled water for 5 min. For sampling of small lesions
or even single cells by microdissection, see chapter 2.1.3
3.2. DOP-PCR Procedure (see Note 1)
An optional Topoisomerase step may be performed (see Note 2).
1. Prepare PCR mix (50-µL volume, see Note 3):
Template
Pretreated cell sample in 20 µL of laser
buffer or up to 100 ng isolated DNA
Primer 6-MW (0.1 nmol/µL, see Note 4) 1.0 µL
10× amplification buffer 5.0 µL
MgCl 2 3.4 µL
dNTP (40 mM) 1.2 µL
Taq Polymerase (5 U/µL) 0.8 µL
H 2 O Fill up to final volume of 50 µL
2. Set up cycler conditions: Start PCR in a thermal cycler in a 50-µL reaction volume using
the following conditions (see Note 5).
Denaturation 10 min at 94°C
Followed by five cycles: 11 min at 94°C
(Low stringency) 11.3 min at 30°C
13 min transition 30–72°C
13 min extension at 72°C
Followed by 35 cycles: 11 min at 94°C
(High stringency) 11 min at 62°C
13 min at 72°C
Final extension 10 min at 72°C
3. Store PCR product at –20°C or continue to next step.
4. Use a 5-µL aliquot of the PCR product to check the DNA fragment size on a 3% agarose
ethidium bromide gel (see also Fig. 1),
5. Determine DNA amounts by fluorometric or photometric measurements.