Nested amplification is performed in the event that the genomic amplification yields are inadequate or extraneous bands are present. A 1:1000 dilution of PCR product is adequate to use as template for all nested amplification reactions. The composition of the dilution buffer is the same as the buffer supplied in the control DNA sample, which is 1 mM Tris HCl pH 8.0, 1 mM EDTA, and 0.01% Tween-20. Use caution during preparation as there is an increased risk of contamination performing nested PCR.
- Prepare the dilution buffer in a quantity sufficient for the number of samples being diluted (e.g. for 48 samples prepare 50 ml).
- Make the dilution by adding 1 µl of PCR product to 999 µl of buffer.
- Use 2 µl of this dilution as template.
- Proceed with amplification following the steps outlined in genomic amplification.
- Follow the amplification with electrophoresis
|
