Primer annealing program

However, the isostabilizing component of the universal annealing buffer offers stability of primer-template duplexes. This enables amplification of different lengths using the same extension time selected for the longest amplicons, without compromising specificity Figure 5.

Figure 5. The universal annealing feature enables co-cycling of short and long amplicons. The same PCR protocol was used for all four targets with the annealing and extension settings indicated.

In this way, different PCR assays can be co-cycled using the same protocol, instead of sequential runs, with the universal annealing buffer Figure 6. This can help you save significant time and simplify PCR protocols, especially when you need different primer sets to detect multiple sequences. Figure 6. Time saving enabled by assay co-cycling and a universal protocol. PCR assays using conventional protocols require sequential runs for amplification of targets of different lengths because of the different primer annealing temperatures and extension steps.

Request a sample where available to try them for free in your experiments. These products can help you save time and simplify PCR protocols by:. Learn the importance of the annealing step in PCR, how to circumvent optimization steps using a specially formulated PCR buffer, and the benefits of a universal annealing temperature enabled by the buffer. Don't have an account? Create Account. Sign in Quick Order. Find out more about these products at www. Secondary structure— Low yields of the expected, annealed product can be caused by secondary structure.

Problematic annealing can often be resolved by slow cooling, as described in Step 3, above. These can be added at the time of oligo synthesis chemical phosphorylation; done by request or anytime thereafter before or after annealing using polynucleotide kinase enzymatic phosphorylation. For a small fee, IDT will anneal your oligos for you, so that you can proceed with your experiments as soon as your oligos arrive.

To request this Duplex Service, go to the Duplex Entry page. Verified by mass spectrometry. Include mixed bases, modifications. Use these programs to calculate T m , identify secondary structure, optimize codon use, select siRNA, and more. All rights reserved. For specific trademark and licensing information, see www.

Order by stock part number ». Primer3 converts concentration of divalent cations to concentration of monovalent cations using formula suggested in the paper Ahsen et al. According to the formula concentration of desoxynucleotide triphosphate [dNTP] must be smaller than concentration of divalent cations. The millimolar concentration of deoxyribonucleotide triphosphate.

This argument is considered only if Concentration of divalent cations is specified. Option for specifying the salt correction formula for the melting temperature calculation. There are three different options available: 1. Schildkraut and Lifson , DOI The default value of Primer3 version 1.

SantaLucia , DOI Owczarzy et al. Option for the table of Nearest-Neighbor thermodynamic parameters and for the method of melting temperature calculation.

Two different tables of thermodynamic parameters are available: Breslauer et al. The nanomolar concentration of annealing oligos in the PCR.

Note that this is not the concentration of oligos in the reaction mix but of those annealing to template. This parameter corresponds to 'c' in Rychlik, Spencer and Rhoads' equation ii Nucleic Acids Research, vol 18, num 21 where a suitable value for a lower initial concentration of template is "empirically determined". The value of this parameter is less than the actual concentration of oligos in the reaction because it is the concentration of annealing oligos, which in turn depends on the amount of template including PCR product in a given cycle.

This concentration increases a great deal during a PCR; fortunately PCR seems quite robust for a variety of oligo melting temperatures. With this option on, the program will automatically retrieve the SNP information contained in template using GenBank accession or GI as template is required and avoid choosing primers within the SNP regions. If the default "Automatic" setting is selected, the program will automatically select the repeat database using the following rules.

If a repeat database is available from the same organism as specified in the "Organism" field by user see above , then that repeat database will be used.

For example, if "Human" is specified, then the human repeat database will be selected. If a repeat database from the same organism is not available, the database from the closest parent of that organism in the taxonomy tree will be selected. For example, the rodent repeat database will be selected if "Mouse" is specified in "Organism" field.

However, no repeat database will be selected if "Gallus gallus" is specified since a repeat database from its taxonomical parents is not available. This option enables our new graphic view which offers much more details for your template and primers. It will replace the current graphic view in the future.

Retrieve recent results Publication Tips for finding specific primers Reset page Save search parameters. 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, bps. If your template is longer than that, you need to use primer range to limit the length i. Range Help Clear.

From To. Forward primer. Reverse primer. Help Optionally enter your pre-designed forward primer. Help Optionally enter your pre-designed reverse primer. Min Max. Min Opt Max Max T m difference. No preference Primer must span an exon-exon junction Primer may not span an exon-exon junction Help This controls whether the primer should span an exon junction on your mRNA template. Min 5' match Min 3' match Max 3' match. Enable search for primer pairs specific to the intended PCR template Help 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.

Automatic User guided No user guidance Help Primer-blast tries to find target-specific primers by placing candidate primers on unique template regions that are not similar to other targets.

Entrez query optional Help You can use a regular entrez query to limit the database search for primer specificity. Primer must have at least 1 2 3 4 5 6 total mismatches to unintended targets, including at least 1 2 3 4 5 6 mismatches within the last 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 bps at the 3' end.

Help This requires at least one primer for a given primer pair to have the specified number of mismatches to unintended targets. Ignore targets that have 1 2 3 4 5 6 7 8 9 or more mismatches to the primer.

Help This is another parameter that can be used to adjust primer specificity stringecy. Show results in a new window Use new graphic view Help This enables our new graphic display that offers enhanced overview for your template and primers. 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 10 50 Help Maximum number of database sequences with unique sequence identifier Blast finds for primer-blast to screen for primer pair specificities.

Help The maximum number of PCR targets amplicons to be shown when designing new primers.