Primers tool

Optionally enter your pre-designed reverse primer. The Tm calculation is controlled by Table of thermodynamic parameters and Salt correction formula under advanced parameters. The default Table of thermodynamic parameters is "SantaLucia " and the default Salt correction formula is "SantaLucia " as recommended by primer3 program. 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. Minimal and maximal number of bases that must anneal to exons at the 5' or 3' side of the junction Help This specifies the minimal number of bases that the primer must anneal to the template at 5' side i.

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. 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.

This specifies the range of total intron length on the corresponding genomic DNA that would separate the forward and revervse primers. 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. 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. 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. This database is recommended if you are not concerned about missing alternate loci or mitochondrion sequences. The database size is limited to M. You can choose to exclude sequences in the selected database from specificity checking if you are not concerned about these. 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.

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.

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. 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 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.

In general, the non-specific targets become less of a concern if their sizes are very large since PCR is much less efficient for larger amplicons.

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.

You just need to be careful when snapping the tray over on, since most primers will bounce turning them the wrong direction. It has a safety gate in the tray to make sure the primer — that is in the priming process is completely separated from the rest of the primers. This is an important safety measure since you can be sure that no primer is going to detonate by accident.

A downside of this product may be the fact that the process may be slower since you have to remove every cartridge by hand once it is primed. Other models simply let go of the case for an easier reload.

After priming, the universal shell holder may tighten the grip, making the removal process slower. There are a few models that already include all the shell holders necessary to begin priming. You should make sure you get a priming tool with these included, or you will have to buy each of them separately. It is no big deal since these are not expensive, but it may be difficult to find some specific sizes. If you are not a fan of this, you can also look for a priming unit with a universal shell holder.

Usually, when you are hand priming a case with a specific shell holder, you can work with a tray or bucket under the primer, so you just have to set the cartridge on the primer, squeeze, release the handle and watch the primed case fall onto the bucket.

But with a universal shell holder, you cannot do this since you get to remove each case by hand each time you prime it.

If you like to practice on a shooting range with your rounds, you may be priming hundreds of cases at a time. Because of this, you would like a comfortable and fast primer. The best hand primers come with an ergonomic handle. You will surely be priming at least a few hundred cases at the time, and your hand will surely cramp up after a while. An ergonomic handle helps you to get a better grip on the tool, reducing the possibility of getting cramps or getting tired because of repeated action.

Also, an ergonomic handle can be useful for more precise priming. Most models do not have a predetermined seating depth, so you will have to feel how deep you want the primer to be set. The better the handle, the more sensitive will it be. This means that you will have less crushed primers to deal with. This may be one of the most important aspects of a hand primer. A single detonated primer can lead to a chain reaction where all the primers o the supply will detonate as well.

Most hand primers nowadays come with a safety gate to separate the primer that is being primed of the rest of the unused primers. You are working with deadly gunpowder. This means that a mistake where you accidentally detonate a single round on a hand primer can cause severe damage. Hand primers are supposed to work specifically as you need them to, but you have to make sure everything is in place and safely secured when using the primer.

Because of the continuous danger you are in when priming, here are a few safety precautions you want to tan into account when working with hand primers. Hearing protection: Detonated primers can make a deafening sound, so you should make sure you have the appropriate hearing protection. Eye protection: You should always have your eyes protected since a minor error can make primer pieces to blow out of the hand priming tool. Proper primer storage: Every primer should be inside their original package when you are not using them.

Also, make sure your store this package in a dry place away from any heat sources. No, if the primers do not have a universal shell holder, you will have to get the shell holders specific to the brand of the tool. Overall both tools get the job done. It mostly depends on your comfortability. If you want to be able to prime cases everywhere, the portable hand primer is a safe bet obviously. On the other hand, mounted hand primers provide increased stability, apart from the fact that the hand primer toll will be right beside your reloading press.

There are certain models in which you can set the desired seating depth. It means that you will never crush a primer again. If by any chance you have a hand primer in which you cannot set any seating depth you will have to feel for yourself. Once you feel the primer set you will have to release.

Because of this, many primers will surely be at a different depth than others. If the press is the same brand as the hand primer you should be able to use the same shell holder.

Sometimes paying a little bit more for greater quality is a good decision, especially when purchasing something you plan to use for a long time. However, we believe that the Frankford Arsenal Perfect Seating Tool is the best hand priming unit here. There is no way you crush the primers, and thanks to the configurable dial that you can set to desired seating depths.

This means that each primer on each case will be set at the same depth as all the other primers. There are 12 shell holders included, one for each one of the most popular cartridge sizes. Also, you can easily store all these holders and the priming tool inside the storage case that is already included when you purchase this model.

The die-cast zinc handle may not be as comfortable, but it is designed to withstand a great force. Also, it comes with a safety gate to isolate the primer. It will be loaded on the cartridge from the primers on the tray to reduce the possibility of tray detonation. We believe that even if the price is a bit too high, it is worth it. This model stands perfect for anyone since it is not at all complicated to use.

It will be money well spent. Joseph Fox writes on a variety of topics ranging from reloading ammunition to gun cleaning. Looking to get the best hand priming tool? This detailed article can help. Top 4 Hand Primer Comparison Table. When reloading your cases, there are many choices you can take before buying new components. Top 4 Hand Priming Tool Reviews You can have the seating depth extremely precise and accurate Includes a case to store the priming tool and the 12 shell holders It is made from resistant and durable materials.

It is one of the most expensive hand priming tools in the market. Safety measures included for reducing the possibility of a tray detonation You can use the same shell holders you use on your RCBS reloading press It is durable and reliable.

Lee Precision Auto Bench Priming Tool Many Lee reloading presses come with a priming unit, but this product is designed to be a full priming station on your workbench. It has a slower removing process than other models because of the universal shell holder.

Safety Measures in the Primer This may be one of the most important aspects of a hand primer. Hearing protection: Detonated primers can make a deafening sound, so you should make sure you have the appropriate hearing protection Eye protection: You should always have your eyes protected since a minor error can make primer pieces to blow out of the hand priming tool.

Is there any difference between mounted hand primers and portable hand primers?