You may not have ROX™ dye in the master mix

The use of a PCR Master Mix that does not contain ROX™ dye (or a similar passive reference) can cause:

• Trailing clusters
• Some or all data is undetermined (There is an "X" instead of a called allele in the AD plot.)
• Diffuse clusters

Solution:

• ROX™ dye is a passive reference dye that improves the precision of the results by compensating for small fluorescent fluctuations, such as bubbles and small well-to-well variations.

Note: The Sequence Detection Software on the 7900HT Fast Real-Time PCR System will not call the alleles when ROX™ dye (or another passive reference) is not present.

• Use Applied Biosystems TaqMan® Genotyping Master Mix or TaqMan® Universal PCR Master Mix, both of which contain ROXÔ passive reference.
• Confirm ROXÔ passive reference is specified in SDS software:

Specify ROX™ passive reference in SDS software