Primers

The Primers tab designs PCR primer pairs for a selected region, checks primers you already have, and keeps your own list of primers, My primers, with where each of them binds on the document.

Melting temperatures use the nearest-neighbour method (SantaLucia 1998, unified parameters) at 500 nM oligo and 50 mM monovalent salt. They are good to about ±2 °C for ordinary PCR primers; adjust for your buffer.

Designing primers for a region

1. Select the region to amplify in either view. It can be a feature (click it in the Features tab) or any range, through the origin of a circle too: the panel then gives the target as the status bar does, `Target

591–20 (30 bp)` on a 600 bp plasmid.

1. Open Primers and click Design primers.
2. Up to ten pairs are listed, best first. Each is headed by its rank, the product size and the Tm difference between the two primers; under that are the forward (→) and reverse (←) primer, each with its Tm and GC content at the right of the line (below it when the panel is narrow).
3. Show draws that pair in both views without touching the document: the two sites as dashed arrows pointing the way each primer reads, and the product between them as a bracket (see Previews). It also selects the product, so the views scroll to it and the status bar gives its length. Hide takes both away again, the drawing and the selection, as does leaving the tab; if you have selected something else in the meantime that selection is yours and is left alone. Hovering a pair shows it for as long as the pointer is there — so three candidates can be compared without annotating anything.
4. Add both annotates the pair as primer_bind features (the primer sequence goes in a /note), so the sites are on the map and saved with the file. Unlike Show, this is an edit: it goes into the History as one step, and one Undo takes both back.
5. Save both keeps the two primers in My primers, named after the document (pUC19 fwd 1, pUC19 rev 1), with the target, Tm and GC content in their notes.

If nothing qualifies, select more flanking sequence, look further from the selection, or loosen the settings.

A design belongs to the sequence it was made on. If you edit the bases, reverse complement, or change the topology or origin, the list is cleared, because the coordinates and oligos no longer fit; select the target and design again. Adding or editing annotations leaves it alone.

Settings

Settings, under the target, sets what a designed primer has to be. Its row sums the main rules up (18–27 nt, Tm 55–65 °C, GC 35–65 %, within 0–200 bp of the target) and says changed once any setting is off its default; open it to see and change them all. Every candidate that breaks one of them is refused, and the same numbers are what Check a primer warns about, so a pasted primer is judged by the rules the designed ones had to pass. They are remembered in this browser, from plasmid to plasmid; Reset to defaults puts them back.

Where to look is measured from the selection's edges: the forward primer lies wholly between the two distances before the start, the reverse primer between the two after the end. A negative number reaches _into_ the selection, so -30–0 for the forward primer puts it on the selection's own first bases — how an ORF is amplified from its start codon. To keep primers well clear of the region, as for sequencing across it, raise the near edge: 50–200 leaves at least 50 bp between each primer and the selection.

Among the candidates that pass, pairs are ranked by a penalty that favours a Tm in the middle of the range, GC in the middle of its range, a GC clamp, little self-complementarity or hairpin, a length near 21 nt and a small Tm difference.

Checking a primer

Paste a sequence under Check a primer. You get its length, Tm, GC content and whether it ends in a GC clamp, plus a warning for each setting it breaks — too short or too long, Tm or GC out of range, a long run of one base, a hairpin, a self-complementary stretch, a 3′ end that pairs with itself — and for a missing GC clamp.

With eight or more bases, the tab also searches the document for binding sites on both strands: the 3′ end must match exactly and up to two mismatches are allowed elsewhere. Every site found is previewed in both views at once, so off-target binding is visible at a glance, and a site's mismatches are marked on its arrow at the bases they fall on. Each site also shows its strand, position and mismatch count in the list; click one to select it, or Add sites as primer_bind to annotate them all, as one step a single Undo takes back. Save to My primers keeps the primer in My primers.

A degenerate primer — one with ambiguity codes, such as NNK for a codon library — is a mix of molecules, and the check says so: how many positions are degenerate and how many molecules the mix holds. Tm and GC content are given as the range over the mix (Tm 58.2–63.9 °C). A setting is broken when all of the mix breaks it, and Part of the mix says when only some does. Past 4,096 molecules (six Ns) the Tm is not given. A code binds wherever it stands for the template's base, so a degenerate primer's sites are the ones some molecule of the mix binds at, as in PCR. Hairpins and dimers are counted on the plain bases.

Use this to check a primer from a paper or an old order against the plasmid you have, and to spot off-target binding.

Note that this search asks whether the whole oligo matches, which is the right question for "is this primer specific to my plasmid" and the wrong one for a cloning primer with a 5′ tail — a restriction site or a Gibson homology arm matches the template nowhere. To amplify with such a primer, use Cloning ▸ PCR, which anneals by the 3′ end and reports the rest as a tail. To design the tailed primers that fuse two or more fragments by PCR, use the Bench's Overlap PCR.

My primers

My primers, at the bottom of the Primers tab, is your own list of primers: a name, the bases and notes for each, with the length and the Tm of the bases beside the name (a degenerate primer shows its length alone). It is kept in this browser's storage, beside the recent files, and like them it never leaves the browser except as a file you download. It is the same list whichever document is open.

Adding primers

• Add primers ▸ One primer: a name, the bases (5′ to 3′, IUPAC codes allowed) and notes. Without a name it is called Primer 1, Primer 2 and so on.
• Add primers ▸ Paste many, then Add pasted, or From a file…, reads a list in any of these shapes: - FASTA: >name notes and the bases on the lines below; - a table, as CSV, tab-separated (what a spreadsheet copies) or semicolon-separated: with a header naming name, sequence and notes columns in any order, or without one, when the column of bases is the sequence, the first other column the name and the rest notes; - one primer per line, the bases alone or after a name; - an Excel workbook (.xlsx), such as an oligo order sheet. Its sheets are tried in order and the first one with a primer in it is read, as a table is; the report says which sheet when there are several. Sheets are not added together. A workbook in which no sheet has a primer is turned away with the names of its sheets. An old .xls workbook or an OpenDocument .ods one is not read: save it as .xlsx or CSV first.

The header need not be the first row: a title or address above it is passed over. Order-sheet headers such as Oligo Name, Sequence Name, Sequence (5' to 3') or Sequence 5'->3' are understood, and a 5' Modification column is added to the notes. 5′-…-3′, spaces between the bases and IDT's modification codes (/5Phos/ACGT…, which go into the notes) are fine. You can also drop the file on the Paste many box; a file is read by what is in it, whatever its name. Lines (or a sheet's rows) with no primer in them are named in the report under the box. A primer already in the list under the same name and with the same bases is left out rather than added twice; the same bases under another name are added.

• Save both under a designed pair, and Save to My primers under Check a primer.
• From this document's primer_bind features adds every primer_bind feature of the open document, and Save to My primers in the feature editor adds one. The oligo is taken from a sequence: note when the feature has one (the Primers tab and SnapGene write one, and it holds a 5′ tail the template does not have), otherwise it is the bases under the feature, read along its strand.

Edit and Delete change one primer; Delete all… asks once more before emptying the list. Past eight primers a filter box finds them by name, notes or bases.

Finding where they bind

Tick Find my primers in _the document_. Every primer of the list is searched on both strands and, on a circular sequence, through the origin, the way PCR anneals a primer: by its 3′ end. The last five bases must match exactly; before them up to two mismatches are allowed (Mismatches sets it, from none to three), and a site needs at least 15 annealed bases (Shortest site sets it, from 10 to 20). The two settings appear under the tick box while it is ticked. What does not anneal at the 5′ end is reported as a tail, so a cloning primer with a restriction site or a homology arm on it is found by the part that binds. Primers shorter than the shortest site are counted but not searched; lower Shortest site to search a short sequencing primer. Under 15 bases a site is more likely to be there by chance, which is why it is not the default.

Each site is listed with its strand, position, name, mismatches and tail, and every site is drawn on both views at once as a preview, mismatches marked on its arrow. The search runs again as you edit.

• Click a site to select the bases it anneals to.
• Add annotates one site as a primer_bind feature, with the whole oligo in a sequence: note and the primer's notes after it; Add all sites as primer_bind annotates every one. Either is one step in the History, so a single Undo takes it back.
• PCR fwd on a forward site, or PCR rev on a reverse one, puts the primer in that slot of Cloning ▸ PCR, under its own name; the list's own PCR fwd and PCR rev buttons do the same for any primer. A line under the sites says what PCR holds, and Open PCR goes there. Editing the bases in PCR by hand drops the name.

At most 200 sites are listed and drawn; a primer inside a repeat can bind many more.

Downloading the list

Download CSV writes primers.csv (name,sequence,notes), which reads back exactly as it was. Download FASTA writes primers.fasta, one >name notes record per primer; a FASTA name ends at the first space, so spaces in a name become underscores. Either can be pasted or loaded back, in this browser or another. Download the list now and then: a browser can clear its storage (see Files and storage).