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Retrieve recent results Publication Tips for finding specific primers

PCR Template

Enter accession, gi, or FASTA sequence (A refseq record is preferred)
Or, upload FASTA file

Range

From To

Forward primer

Reverse primer

Primer Parameters Use my own forward primer (5'->3' on plus strand)

Use my own reverse primer (5'->3' on minus strand)

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

Min 5' match Min 3' match Max 3' match

Minimal and maximal number of bases that must anneal to exons at the 5' or 3' side of the junction Help

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

Search mode

Database

Refseq mRNA:
    This contains mRNA only from NCBI's Reference Sequence collection

Refseq representative genomes:
    This database contains NCBI RefSeq Reference and Representative genomes across broad taxonomy groups including eukaryotes, bacteria, archaea, viruses and viroids. These genomes are among the best quality genomes available at NCBI. This database contains minimum redundancy in genome representation. For the eukaryotes, only one genome is included per species (However, alternate loci of eukaryotic genomes are included where applicable). For other species, genomes from diverse isolates of the same species may be included. Mitochondrion genomes are included where applicable.

core_nt:
    It is the same as nt except that it does not have the eukaryotic chromosomal sequences that are part of NCBI genome assemblies. The search speed is much faster than nt. We highly recommend using it over nt.

Refseq RNA:
    This contains all RNA entries from NCBI's Reference Sequence collection

Genomes for selected eukaryotic organisms (primary assemblies only):
   These are Refseq representative genomes from primary chromosome assemblies (i.e., no alternate loci) for many eukaryotic organisms. Mitochondrion and plastid genomes are also included where applicable.

Although sequences in this database are completely covered by the Refseq representative genomes database, it does not contain the alternate loci and thus avoids sequence redundancy introduced by including alternate loci. This database is recommended if you are not considering variations represented by alternate loci.

Custom:
   You can use your own sequences including nucleotide accessions, genome assembly accessions (such as GCF_000001635.27) or FASTA sequences as a search database. A maximum of 20 assembly accessions are allowed. FASTA sequences are limited to 300M. Note that the organism field is ignored for custom database.

Enter sequence accession, FASTA sequence or assembly accession Clear

Or, upload file:

Exclusion

Exclude predicted Refseq transcripts (accession with XM, XR prefix) Exclude uncultured/environmental sample sequences

Organism

Enter an organism name (or organism group name such as enterobacteriaceae, rodents), taxonomy id or select from the suggestion list as you type. Help

Entrez query (optional)

Primer specificity stringency

Primer must have at least 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.

Max target amplicon size

Allow splice variants

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

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

Advanced parameters

Primer Pair Specificity Checking Parameters Max number of sequences returned by Blast

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)

Max targets per sequence

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 Use Thermodynamic Template Alignment (warning: search may be very slow with this option on)

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

Minimal number of nucleotides that the left or the right primer must have at the 5' or 3' side of the junctions

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

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