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updates. High fidelity qPCR Kristie Nybo, Ph. D. BioTechniques,
Vol. 55, No. 4, October 2013, pp. 171–173 Full Text (PDF)
Molecular Biology Techniques Q&A  Why doesn't my qPCR work with
a high fidelity enzyme and SYBR green? (Thread 29684)  Q: I've
always used SYBR Green or HYBProbe master mix kits for my qPCR
reactions. But for my current project, I need to sequence the
products following qPCR. My advisor says that a high fidelity
enzyme should be used here since we will be sequencing the
products, which means I can't use the master mixes.  I purchased
10,000× SYBR Green along with Phusion high fidelity polymerase
and have been trying to use this combination for qPCR, but
haven't been getting any amplification. The Phusion enzyme comes
with its own 5× buffer that I add to my mix along with 0.2 mM of
each dNTP, 0.5 µM of each primer, and 0.4 U polymerase in a 20
µl reaction. I use SYBR Green at a final concentration of 1×.
The primers work in qPC