Primers and Probes can be
designed using Primer Express software as described in the Primer Express
User Bulletin (P/N 4317594).
**PROBES:
_ Keep the G-C content in
the 20 to 80% range.
_ Avoid runs of an
identical nucleotide. This is especially true for guanine, where runs of four
or more Gs should be avoided.
_ **Select the strand that
gives the probe more Cs than Gs (Primer Express does not check this for
you).
_ **Do not put a G on the 5´ end.
_ **For single-probe assays, the melting
temperature (Tm
) should be
68 to 70 °C when using Primer Express software (10 °C higher than primer Tm). For Allelic Discrimination Tm of the probe
should be 65–67 °C and no more than 1° Tm difference between the two probes
_ **Both probes should be on the same strand (AD).
_**For
gene expression, consider placing probe over exon-exon junction to reduce
amplification of contaminating genomic DNA.
_Use MGB specific document
to design MGB probes (unique Tm calculator).
**PRIMERS:
_ **Choose the primers after the probe.
_ Design the primers as
close as possible to the probe without
overlapping the probe.
_ Keep the G-C content in
the 20 to 80% range.
_ Avoid runs of an
identical nucleotide. This is especially true for
guanine, where runs of
four or more Gs should be avoided.
_ **The T m of each primer should be 58 to 60 °C.
_ **The five nucleotides at the 3´ end of each
primer should have no more than two G and/or C bases.
** unique to
Probes
** unique to Primers
FOR REFERENCE USE ONLY