AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Instrukcja Użytkownika Pobierz pdf (Strona 21)

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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 and Probes

14 Primer Express Software 3.0 Getting Started Guide

Notes

Figure 4. Primer Probe Test Tool dialog box

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

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

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

5. Paste (Ctrl+V) the putative sequence in the Probe 1 field. The Primer Probe Test

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

1 field.

6. If the Tm is not between 68 °C to 70 °C, highlight a section of the sequence to view

the corresponding Tm, %GC, and oligonucleotide length. Once the highlighted

region results in the desired Tm, click on Trim to delete the non-highlighted bases.

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

guidelines, refer to Primer Express Software Version 3.0 Online Help):

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

• Probe Length – 13 to 25 bases (13 to 30 bases if using conventional

TaqMan probes)

• Tm – 68 °C to 70 °C.

• % GC – 30% to 80%.

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AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Instrukcja Użytkownika Strona 21