Primer designing tool

The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. Enter the PCR template here (multiple templates are currently not supported). It is highly recommended to use refseq accession or GI (rather than the raw DNA sequence) whenever possible as this allows Primer-BLAST to better identify the template and thus perform better primer specificity checking.
A template is not required if both forward and reverse primers are entered below.
The template length is limited to 50,000 bps. If your template is longer than that, you need to use primer range to limit the length (i.e., set forward primer "From" and reverse primer "To" fields but leave forward primer "To" and reverse primer "From" fields empty). Or, upload FASTA file Enter the position ranges if you want the primers to be located on the specific sites. The positions refer to the base numbers on the plus strand of your template (i.e., the "From" position should always be smaller than the "To" position for a given primer). Partial ranges are allowed. For example, if you want the PCR product to be located between position 100 and position 1000 on the template, you can set forward primer "From" to 100 and reverse primer "To" to 1000 (but leave the forward primer "To" and reverse primer "From" empty).
Note that the position range of forward primer may not overlap with that of reverse primer. Optionally enter your pre-designed forward primer. Always use the actual primer sequence (i.e., 5'->3' on plus strand of the template). Please enter the primer sequence only (No any other characters are allowed). Use my own reverse primer (5'->3' on minus strand) Optionally enter your pre-designed reverse primer. Always use the actual primer sequence (i.e., 5'->3' on minus strand of the template). Please enter the primer sequence only (No any other characters are allowed). PCR product size Exon/intron selection A refseq mRNA sequence as PCR template input is required for options in the section A refseq mRNA sequence (for example an entrez sequence record that has accession starting with NM_) allows the program to properly identify the corrsponding genomic DNA and thus find correct exon/intron boundaries. Exon junction span This controls whether the primer should span an exon junction on your mRNA template. The option "Primer must span an exon-exon junction" will direct the program to return at least one primer (within a given primer pair) that spans an exon-exon junction. This is useful for limiting the amplification only to mRNA. You can also exclude such primers if you want to amplify mRNA as well as the corresponding genomic DNA. 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 This specifies the minimal number of bases that the primer must anneal to the template at 5' side (i.e., toward start of the primer) or 3' side (i.e., toward end of the primer) of the exon-exon junction. Annealing to both exons is necessary as this ensures annealing to the exon-exon junction region but not either exon alone. Note that this option is effective only if you select "Primer must span an exon-exon junction" for "Exon junction span" option. Intron inclusion Primer pair must be separated by at least one intron on the corresponding genomic DNA With this option on, the program will try to find primer pairs that are separated by at least one intron on the corresponding genomic DNA using mRNA-genomic DNA alignment from NCBI. This makes it easy to distinguish between amplification from mRNA and genomic DNA as the product from the latter is longer due to presence of an intron. Intron length range This specifies the range of total intron length on the corresponding genomic DNA that would separate the forward and revervse primers. 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 With this option on, the program will search the primers against the selected database and determine whether a primer pair can generate a PCR product on any targets in the database based on their matches to the targets and their orientations. The program will return, if possible, only primer pairs that do not generate a valid PCR product on unintended sequences and are therefore specific to the intended template. Note that the specificity is checked not only for the forward-reverse primer pair, but also for forward-forward as well as reverse-reverse primer pairs. Search mode Primer-blast tries to find target-specific primers by placing candidate primers on unique template regions that are not similar to other targets. However, in some cases, primer-blast cannot determine if a database sequence is an intended target or not, thus the user guidance might be helpful (For example, when your template is a polymorphic form or a partial region of an entry in the search database, or when the database such as the nr contains redundant entries of your template). The "Automatic" option will ask for user guidance only when the program does not find sufficient unique template regions while the "User guided" option will always ask for user guidance if your template shows high similarity to any other database sequences. Refseq mRNA:
&nbsp&nbsp&nbsp This contains mRNA only from NCBI's Reference Sequence collection
Refseq representative genomes:
&nbsp&nbsp&nbsp 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:
&nbsp&nbsp&nbsp 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:
&nbsp&nbsp&nbsp This contains all RNA entries from NCBI's Reference Sequence collection

Genomes for selected eukaryotic organisms (primary assemblies only):
&nbsp&nbsp&nbspThese 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: &nbsp&nbsp&nbspYou 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 ( Use file upload below for large input ) Clear
Or, upload file: You can choose to exclude sequences in the selected database from specificity checking if you are not concerned about these. There are a large number of predicted Refseq transcripts in the Refseq mRNA, Refseq RNA and nr database. There are also many uncultured/environmental sample sequencesare in the nr database.

Enter an organism name (or organism group name such as enterobacteriaceae, rodents), taxonomy id or select from the suggestion list as you type. This will limit the primer specificity checking to the specified organism. It is strongly recommended that you always specify the organism if you are amplifying DNA from a specific organism (because searching all organisms will be much slower and off-target priming from other organisms is irrelevant). Click on "Add more organisms" label if you want to restrict to multiple organisms (enter only one organism in each input box). You can use a regular entrez query to limit the database search for primer specificity. For example, enter a GenBank accession number to limit search to that particular sequence only (Caution: this means the primer specificity will NOT be checked against any other sequences except the specified one). 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. This requires at least one primer (for a given primer pair) to have the specified number of mismatches to unintended targets. The larger the mismatches (especially those toward 3' end) are between primers and the unintended targets, the more specific the primer pair is to your template (i.e., it will be more difficult to anneal to unintended targets). However, specifying a larger mismatch value may make it more difficult to find such specific primers. Try to lower the mismatch value in such case. Ignore targets that have or more mismatches to the primer. This is another parameter that can be used to adjust primer specificity stringecy. If the total number of mismatches between target and at least one primer (for a given primer pair) is equal to or more than the specified number (regardless of the mismatch locations), then any such targets will be ignored for primer specificity check. For examaple, if you are only interested in targets that perfectly match the primers, you can set the value to 1. You can also lower the E value (see advanced parameters) in such case to speed up the search as the high default E value is not necessary for detecting targets with few mismatches to primers.
Additionally this program has limit detecting targets that are too different from the primers...it will detect targets that have up to 35% mismatches to the primer sequences (i.e., a total of 7 mismatches for a 20-mer).
You may need to choose more sensitive blast parameters (under advance parameters) if you want to detect targets with a higher number of mismatches than default. Allow primer to amplify mRNA splice variants (requires refseq mRNA sequence as PCR template input) If enabled, this program will NOT exclude the primer pairs that can amplify one or more mRNA splice variants from the same gene as your PCR template, thus making primers gene-specific rather than transcript-specific (Note that it is NOT intended to generate primers that will amplify all variants. It only means that the primers may amplify one or more other slice variants, in addition to the one you have specified). Enabling this option will make it much easier to find gene-specific primers since there is no need to distinguish between splice variants. This option requires you to enter a refseq mRNA accession or gi or fasta sequence as PCR template input because other type of input may not allow the program to properly interpret the result. Maximum number of database sequences (with unique sequence identifier) Blast finds for primer-blast to screen for primer pair specificities. Note that the actual number of similarity regions (or the number of hits) may be much larger than this (for example, there may be a large number of hits on a single target sequence such as a chromosome). Choose a higher value if you need to perform more stringent search. Expected number of chance matches in a random model. A higher E value should be used if you want more stringent specificity checking (i.e., to identify targets that have more mismatches to the primers, in addition to the perfectly matched targets). On the other hand, a lower E value is recommended if you are only interested in perfect or nearly perfect matches as this will significatly shorten the search time. Use Thermodynamic Template Alignment (warning: search may be very slow with this option on) The option "Use Thermodynamic Oligo Alignment" instructs Primer3 to use thermodynamic alignment models (instead of old traditional secondary structure alignment) for calculating the propensity of oligos to form hairpins and dimers while the option "Use Thermodynamic Template Alignment" instructs Primer3 to use thermodynamic alignment models (instead of old traditional secondary structure alignment) for calculating the propensity of oligos to anneal to undesired sites in the template sequence. TH: Max Template Mispriming Primer (For thermodynamic alignment model only) TH: Max Self Complementarity (For thermodynamic alignment model only) TH: Max Pair Complementarity (For thermodynamic alignment model only) TH: Max Primer Hairpin (For thermodynamic alignment model only) E.g. 401,7 68,3 forbids selection of primers in the 7 bases starting at 401 and the 3 bases at 68. Or mark the source sequence with < and >: e.g. ...ATCT<CCCC>TCAT... forbids primers in the central CCCC. Overlap junctions Enter a list of space separated nucleotide positions. This requires that the left or the right primers to span a junction that is just 3' of any such positions. For example, entering "50 100" would mean that the left or the right primers must span the junction between nucleotide position 50 and 51 or the junction between position 100 and 101 (counting from 5' to 3'). You can also specify in the fields below the minimal number of nucleotides that the left or the right primer must have on either side of the junctions. This option is useful if you want a primer to a span specific junction on the template. Note that this option cannot be used in association with the "Exon/intron selection" options above. The millimolar concentration of divalent salt cations (usually MgCl2+ in the PCR). Primer3 converts concentration of divalent cations to concentration of monovalent cations using formula suggested in the paper Ahsen et al., 2001 . [Monovalent cations] = [Monovalent cations] + 120*(v([divalent cations] - [dNTP])). According to the formula concentration of desoxynucleotide triphosphate [dNTP] must be smaller than concentration of divalent cations. The concentration of dNTPs is included to the formula beacause of some magnesium is bound by the dNTP. Attained concentration of monovalent cations is used to calculate oligo/primer melting temperature. See Concentration of dNTPs to specify the concentration of dNTPs. Concentration of dNTPs The millimolar concentration of deoxyribonucleotide triphosphate. This argument is considered only if Concentration of divalent cations is specified. Salt correction formula