Primer designing tool

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PCR Template

(A refseq record is preferred) [?] Clear Enter accession, gi, or FASTA sequence

Or, upload FASTA file

Range

From To

Forward primer

Reverse primer

[?] Clear

Primer Parameters

Use my own forward primer (5'->3' on plus strand) [?] Clear

Use my own reverse primer (5'->3' on minus strand) [?] Clear

PCR product size Min Max

# of primers to return

Primer melting temperatures (T_m) Min Opt Max Max T_m difference [?]

Exon/intron selection

A refseq mRNA sequence as PCR template input is required for options in the section [?]

Exon junction span [?]

Exon junction match Exon at 5' side Exon at 3' side

Minimal number of bases that must anneal to exons at the 5' or 3' side of the junction [?]

Intron inclusion Primer pair must be separated by at least one intron on the corresponding genomic DNA [?]

Intron length range Min Max [?]

Note: Parameter values that differ from the default are highlighted in yellow

Primer Pair Specificity Checking Parameters

Specificity check Enable search for primer pairs specific to the intended PCR template [?]

Database [?]

Clear
Enter accession number, gi, or FASTA sequence
Or, upload file:

Organism

Enter an organism name, taxonomy id or select from the suggestion list as you type. [?]

Exclusion (opional)

Exclude predicted Refseq transcripts (accession with XM, XR prefix) Exclude uncultured/environmental sample sequences Use Thermodynamic Template Alignment (warning: search may be very slow with this option on) [?]

Entrez query (optional) [?]

Primer must have at least Primer specificity stringency total mismatches to unintended targets, including
at least mismatches within the last bps at the 3' end. [?] Ignore targets that have or more mismatches to the primer. [?]

Misprimed product size deviation [?]

Splice variant handling

Allow primer to amplify mRNA splice variants (requires refseq mRNA sequence as PCR template input) [?]

Get Primer

Show results in a new window Use new graphic view [?]

Advanced parameters Note: Parameter values that differ from the default are highlighted in yellow

Primer Pair Specificity Checking Parameters

Max number of Blast target sequences [?]

Blast expect (E) value [?]

Blast word size [?]

Max primer pairs to screen [?]

Max targets to show (for designing new primers) [?]

Max targets to show (for pre-designed primers) [?]

Primer Parameters PCR Product Tm

Min Opt Max

Primer Size Min Opt Max

Primer GC content (%) Min Max

GC clamp [?]

Max Poly-X [?]

Max 3' Stability [?]

Max GC in primer 3' end

[?]

Secondary Structure Alignment Methods

Use Thermodynamic Oligo Alignment [?]

TH: Max Template Mispriming Primer Pair (For thermodynamic alignment model only)

TH: Max Self Complementarity Any 3' (For thermodynamic alignment model only)

TH: Max Pair Complementarity Any 3' (For thermodynamic alignment model only)

TH: Max Primer Hairpin (For thermodynamic alignment model only)

Max Template Mispriming Primer Pair (For old secondary structure alignment model only)

Max Self Complementarity

Any 3'

(For old secondary structure alignment model only)

Max Pair Complementarity Any 3' (For old secondary structure alignment model only)

Excluded regions [?]

Overlap junctions [?]

5' side overlaps 3' side overlaps

Concentration of monovalent cations [?]

Concentration of divalent cations [?]

Concentration of dNTPs [?]

Salt correction formula [?]

Table of thermodynamic parameters [?]

Annealing Oligo Concentration [?]

SNP handling Primer binding site may not contain known SNP [?]

Repeat filter [?] Avoid repeat region for primer selection by filtering with repeat database

Low complexity filter Avoid low complexity region for primer selection [?]

Internal hybridization oligo parameters Hybridization oligo

Pick internal hybridization oligo

Hyb Oligo Size

Min Opt Max

Hyb Oligo tm Min Opt Max

Hyb Oligo GC% Min Opt Max

Get Primer

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Text Only Options