Biology Reference
In-Depth Information
10. For an optimal real-time PCR effi ciency of 2.00 per cycle, the
amplicon length should not exceed 150 bp.
11. When selecting the appropriate HKG, the following guidelines
should be considered:
The expression level of the HKG should be approximately
the same Ct value, whereas the Ct values of the target genes
can differ. Ideally, the change in the mean (the sample mean
minus the control mean) for HKGs should be between 0.5 and
0.9 and should not exceed 1. If the change in the mean is
greater than 1.5, it cannot be accepted as “not regulated”;
therefore, it cannot be used as an appropriate HKG for further
calculations.
12. For the separation of shorter fragments, a mixture of
HR-agarose and NuSieve agarose is recommended. BMA
NuSieve 3:1 agarose is suitable for the fi ne separation of small
(less than 1,000 bp) DNA fragments because of its high melt-
ing point (<90 °C) and low background fl uorescence. The
NuSieve agarose alone is very brittle and is diffi cult to handle;
therefore, it is recommended to mix the NuSieve with normal
agarose at a ratio of 1:1.
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