Preparation of PCR Material

The amplification conditions described are those that have given reproducible results. However, optimum amplification conditions (e.g., Mg2+, oligonu-cleotide, and dNTP concentrations) should be established empirically for each oligonucleotide. When designing the X-dT oligonucleotides (e.g., Notl dT), it was found that oligonucleotides with an X component of 15-36 bases gave good amplification, but it was found that shorter oligonucleotides generally gave a lower yield (see Note 1).

1. Amplification of driver. Add 1 ng of material previously amplified by NotI dT to a 400 pL PCR reaction using NotI 24 as the amplification oligonucleotide. Ther mal profile, 1 min 20 s at 94°C, 1 min 20 s at 42°C, 2 min 30 s at 72°C, x 25 cycles.

2. Amplification of tracer. Add 1 ng of material previously amplified by NotI dT to a 200 ||L PCR reaction using T7dT as the amplification oligonucleotide. Thermal profile: 1 min at 94°C, 1 min at 42°C, 2 min at 72°C, x 25 cycles.

3. Purify driver-amplified material using a Qiagen Tip-20 following the manufacturer's protocol. However, to avoid losses in yield because of the small size of the PCR products, ensure that the wash buffer QB1 given above replaces the original QB buffer supplied by the manufacturer (see Note 2).

4. Concentrate the eluted material (final vol 0.8 mL) by precipitating with 80 ||L 3 M NaAc and 650 ||L isopropanol. Chill on wet ice for 20 min, and spin full speed in a refrigerated centrifuge for 15 min. Wash pellet with 70% ethanol, dry, and resuspend in 50 ||L HE (see Note 3).

5. Purify tracer-amplified material by a QIAspin PCR purification column using the standard manufacturer's protocol.

6. Quantitate recovery of PCR material by gel electrophoresis, by comparing recovered material to a dilution series of a well-characterized PolyA PCR sample.

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