Power SYBR Green PCR Master Mix and RT-PCR Protocol (PN ...

Chapter 2
PCR
Designing Custom Target Sequences for
Quantitation
Overview
To design custom primers and identify target sequences for
amplification and quantitation:
Step
Action
1. Install Primer Express ® Software
2. Identify Target Sequence and Amplicon Size
3. Design Primers
Identifying Target
Sequence and
Amplicon Size
Designing
Primers
A target template is DNA, a plasmid containing the nucleotide
sequence of interest, genomic DNA, cDNA, or RNA.
Design primers to amplify short segments of a target (DNA, cDNA,
or RNA) within the target sequence. These short segments are called
amplicons. Shorter amplicons work most efficiently, 50- to 150-bp
sequences yielding the most consistent results.
Design primers using Primer Express Software as described in the
Primer Express ® Version 3.0 Getting Started Guide (PN 4362460).
Note: For more information on design guidelines, refer to the Primer
Express ® Software Version 3.0 Online Help.
General Guidelines
• Do not overlap primer and probe sequences. The optimal primer
length is 20 bases.
• Keep the GC content in the 30 to 80% range.
• Avoid runs of identical nucleotides. If repeats are present, there
must be fewer than four consecutive G residues.
• Keep the T m between 58 to 60 °C.
• Make sure the last five nucleotides at the 3´ end contain no more
than two G and/or C bases.
2-2 Power SYBR
DRAFT
® Green PCR Master Mix Protocol
September 29, 2005 1:24 pm, 2_Chapter.fm