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Analyze a lo-pL aliquot on an agaroseor PAGE gel; stain with ethidium bromide. 7. Reliable PCR Experiments In clinical diagnosis, reliability is the most important criterion for the application of a test. The extreme sensitivity of the PCR requires high standards for the conditions of operation. Even minor contaminations with foreign DNA will render results uncertain. With this caveat in mind, some precautions are necessary to avert false positive results (30). l l l l Separate all PCR materials and equipment from processes Involved m DNA tsolatton.

A well-balanced ratio of the primer concentration is crucial. On the otherhand,theratio of single-strandedto double-stranded product will be inferior at primer concentrations that are too high. 7 as compared to amplifications with stoichiometrlc concentrations. Single-stranded DNAcan be recovered by affinity chromatographic purification after amplification with biotinylated primers (27). In an elegant experiment (81), aphage promoter was attached 5’ onto at least one of the PCR primers. The amplified segments were transcribed into RNA and sequenced with reverse transcriptase.

5 fM. Primers and enzyme are used in large excess (1 uA4 and 1 I-N, respectively). 8 1,s 1,4 1,2 l0,0 0 1 10 I 20 cycles I 30 I 40 Fig 2 Typlcal PCR Changes In efficiency and product yield Graphs were calculated on the basis of 1 @4 of each primer spanning a stretch of 300 bp, 200 p&I each dNTP, 2 U enzyme 1 nM (processing 10 templates/cycle, estimated enzyme degradation S%/cycle), and 200-ng template (0 05 aMo1 single copy gene) Volume of 100 pL this reactron will be close to the theoretical value of 2, which means that the sequence is doubled in every cycle.

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