AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Manual de usuario descargar pdf (Pagina 32)

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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

Primer Express Software 3.0 Getting Started Guide 25

Notes

• 3′ end – Make sure the last five nucleotides at the 3′ end contain no more

than two G + C residues.

Avoid the following motifs:

• Repeating oligonucleotides – Avoid runs of identical nucleotides. If

repeats are present, there must be fewer than four consecutive G residues.

For secondary structure design considerations, see Primer Express Software Version

3.0 Online Help.

Note: If you cannot achieve the recommended Tm, you can design using the

complementary sequence. For more information, see Primer Express Software Version

3.0 Online Help.

To design the Reverse Primer:

1. In the sequence tab, select a putative reverse primer sequence region (containing at

least 25 bases).

IMPORTANT! The Primer Probe Test Tool eliminates non-ATCG bases. Before copying

a sequence, change any non-ATCG bases, or select a different region of the sequence.

2. Select Edit > Copy Complement.

3. On the Primer Probe Test Tool dialog box, paste (Ctrl+V) the primer sequence into

the Rev Primer field. The Primer Probe Test Tool displays the Tm, %GC, and the

oligonucleotide length to the right of the Rev Primer field.

4. 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. Once the highlighted

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

Be sure to keep the above guidelines in mind.

Note that you can further customize your primer by editing the default parameter values

found under the Parameters tab. For more information on editing parameters, see Primer

Express 3.0 Software Online Help.

Saving Primer

Sequences

Copy and paste the primer and probe sequences into a text document, then save for

future reference.

Ordering Primers To order your selected primer/probe set, refer to Chapter 4, “Ordering Primers and

Probes.”

IMPORTANT! Before running your samples, Applied Biosystems recommends that you

run control samples to verify the performance of the selected primers and probes.

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AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Manual de usuario Pagina 32