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Troubleshooting for PCR and multiplex PCR

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1801

 

Troubleshooting discussion is based on the PCR protocol as described in the table below. All reactions are run for 30 cycles.

 

COMPONENT

VOLUME

FINALCONCENTRATION

1.autoclaved ultra-filtered water (pH 7.0)

20.7µL

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2.10x PCR Buffeundefined

2.5µL

1x

3.dNTPs mix (25 mM each nucleotide)

0.2µL

200 µM (each nucleotide)

4.primer mix (25 pmoles/µL each primer)

0.4µL

0.4 µM (each primer)

5.Taq DNA polymerase (native enzyme)

0.2µL

1 Unit/25 µL

6.genomic DNA template (100 ng/µL)

1.0µL

100 ng/25 µL

 

 

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