AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Manuel d'utilisateur télécharger pdf (Page 31)

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December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm

Chapter 2 Designing Primers and Probes for Quantification Assays

Manually Designing Primers for SYBR

Green Dye Assays

24 Primer Express Software 3.0 Getting Started Guide

Notes

Figure 7. Primer Probe Test Tool dialog box

4. From the Document Type drop down menu, select the desired document type.

Verify that the Parameter field is set to Default. For more information about

changing parameters, see Primer Express Software Version 3.0 Online Help.

5. Paste (Ctrl+V) the annotated sequence in the Fwd Primer field. The software

displays the Tm, %GC, and the oligonucleotide length to the right of the Fwd

Primer field.

6. If the Tm is not between 58 °C to 60 °C, highlight a section of the sequence to view

the corresponding Tm, %GC, and oligonucleotide length as if those highlighted

bases were deleted. Once the highlighted region results in the desired Tm, click on

Trim to delete the highlighted bases.

Ensure the following guidelines are met (for more information on design

guidelines, refer to Primer Express Software Online Help):

• Amplicon Length – 50 to 150 bases for optimum PCR efficiency.

• Optimal Primer Length – 20 bases. Do not overlap primer and probe

sequences.

• Tm – 58 °C to 60 °C (Optimal Tm – 59 °C).

• % GC – 30% to 80%.

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AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Manuel d'utilisateur Page 31