Biology Reference
In-Depth Information
curve should be as close to 1 as possible. Second, PCR effi -
ciency for each primer pairs should be as close to 100 % as pos-
sible, equivalent to a slope of −3.32. A good reaction should
have an effi ciency between 90 % and 110 %, which corresponds
to a slope of between −3.58 and −3.10. Third, disassociation
analysis is recommended when using SYBR green dye as a
detector, it is used to determine the specifi city of qPCR. For
good qPCR with high specifi city, every sample, including the
standard DNAs but not the negative control, should show one
single sharp overlapped peak in the dissociation analysis (also
called a melting curve). Finally, to ensure the precision of the
qPCR reaction, each sample should be run in three replicates
and have a standard deviation of Cq less than 0.167.
6. The plasmid pTG3602 [
16
], which contains the whole Ad5
genome, is frequently used as a standard DNA in our qPCR
experiments. It can be used for any primers that target the Ad
genome. However, any other plasmids or purifi ed viral DNA
can serve as standard DNA. The copy number can be calcu-
lated using a web tool (
http://www.thermoscientifi cbio.
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