AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Instrukcja Użytkownika Pobierz pdf (Strona 24)

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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

Primer Express Software 3.0 Getting Started Guide 17

Notes

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 as if those highlighted

bases were deleted. 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.

• 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%.

• 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.

To design the Reverse Primer:

1. In the sequence tab, select a sequence (at least 25 bases) to the right of the probe.

The sequence should be as close to the probe without overlapping it.

2. Select Edit > Copy Complement.

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.

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

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

oligonucleotide length to the right of the Fwd 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 and probe set 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

and Probe

Sequences

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

future reference.

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