Free YouTube Downloader. IObit Uninstaller. Internet Download Manager. Advanced SystemCare Free. VLC Media Player. MacX YouTube Downloader. Microsoft Office YTD Video Downloader. Adobe Photoshop CC. VirtualDJ Avast Free Security. WhatsApp Messenger. Other combinations of proofreading and non-proofreading polymerases have been used successfully for many applications. The buffer listed below works well with Tth and Vent, but not with others.
If you are interested in using other polymerases make sure that you use compatible buffers. 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.
This enables our new graphic display that offers enhanced overview for your template and primers. 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. 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. The minimal number of contiguous nucleotide base matches between the query sequence and the target sequence that is needed for BLAST to detect the targets.
Set a lower value if you need to find target sequences with more mismatches to your primers. However this will increase the search time. The maximum number of candidate primer pairs to screen in order to find specific primer pairs The candidate primers are generated by primer3 program.
Increasing this number can increase the chance of finding a specific primer pair but the process will take longer. The maximum number of PCR targets amplicons to be shown when designing new primers. The maximum number of PCR targets amplicons to be shown when checking specificity for pre-designed primers.
The maximum number of PCR targets amplicons to be found on any single sequence in the search database. The number of consecutive Gs and Cs at the 3' end of both the left and right primer. The maximum stability for the last five 3' bases of a left or right primer.
Bigger numbers mean more stable 3' ends. The maximum number of Gs or Cs allowed in the last five 3' bases of a left or right primer. 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.
Or mark the source sequence with : e. 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 " would mean that the left or the right primers must span the junction between nucleotide position 50 and 51 or the junction between position and 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. 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 Help The millimolar concentration of salt usually KCl in the PCR.
Primer3 uses this argument to calculate oligo melting temperatures. 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. The first version of the software appeared on the market in It went through several modifications, and the last one, the change from version 6 to 7, was the most comprehensive. Oligo search protocols scoring system may be customized in detail, so you may optimize the results according to your specific needs.
Oligo runs on Macintosh and Windows and it may be downloaded from this site.
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