Keep a Primer Library
A primer you designed once is a primer you will want again. Saving it makes it a sequence of its own in the project, registered as an oligonucleotide with an OL- identifier, so whoever amplifies that region next finds it before designing a new one and can order more from its page.
Placeholder: Screenshot Coming Soon Primer Design open on a plasmid, with the line 2 primers in your library bind here and a Choose from library button above the designed pairs, and the Primer Library dialog box open over it, each row showing a primer's name, its OL identifier, the bases it covers, its Tm and Forward or Reverse.
Saving a Primer Pair
Save to library sits on every pair that Primer Design returns, beside Copy forward, Copy reverse and Add as annotation. Primer and CRISPR Design covers the search itself.
It saves the forward and the reverse primer as two sequences, in the same folder as the sequence you designed them on, and a message names the identifiers they were given, such as OL-0007 and OL-0008. Each one keeps:
| Kept on the primer | What it holds |
|---|---|
| Name | The template's name, the direction and the first base it binds, such as pUC19 forward primer 413. Rename it like any sequence |
| Identifier | The next free OL- number |
| Tm and GC | The values Primer Design showed for it |
| Binding site | The sequence it was designed on, the strand it reads along, and its first and last base |
Once both primers of a pair are saved, the button reads In your library. A pair that shares a primer with one you saved earlier saves only the primer that is new.
An oligonucleotide you register by hand with Register Item is in the library too. The Sequence Registry covers that form.
Saving needs edit access to the project. A view-only member sees the library and can copy any primer in it.
Finding Primers That Bind
Open Design Primers on any DNA sequence. When primers in the library bind it, Primer Design opens with a line such as 3 primers in your library bind here above the results.
Choose from library on that line opens the Primer Library dialog box, with a search box that finds a primer by name or identifier. Each row gives the primer's name and identifier, the bases it covers counted from 1, its Tm on this sequence, and Forward or Reverse.
A primer binds when the last 15 bases at its 3' end match the sequence exactly, on either strand. Three things follow from that rule:
- On a circular sequence, a primer lying across the origin is found, and its position reads from the higher number to the lower, such as
2993-12. - The match is followed back toward the 5' end for as long as the bases agree. That stretch is the part that anneals, and the Tm on the row is calculated for it.
- Bases left over at the 5' end, such as a restriction site you added, are counted as a tail, and the row says how long it is, such as
6-base 5' tail.
A primer that binds in two places gets a row for each.
Pick one forward primer and one reverse primer. Picking a second primer that reads the same way replaces the first. The bottom of the dialog box gives the product size for the two you picked, and Use as a pair shows them in Primer Design as Pair from your library, with the product size, the difference in Tm, and the score and warnings a designed pair gets.
The pair carries the same copy and annotation buttons as a designed pair. Remove pair takes it back out of the results, and Choose from library opens the dialog box again with the two primers still picked.
On a linear sequence the reverse primer has to bind downstream of the forward one. Pick two that face away from each other and the dialog box says they make no product here.
Browsing the Library
Primers, under Show in View on the sequences list, narrows the list to registered oligonucleotides, with a count beside it. It lists the primers in the folder you have open and in every folder inside it, so from the top level it shows the whole library. It appears once the project has one. Choose it again to see every sequence.
Opening a primer shows Forward primer or Reverse primer beside its name. Click it to see:
- The sequence it binds and the bases it covers there. Click the name to open that sequence
- Its Tm and GC
- Its scale and purification, when the primer records them
Scale and purification are fields of the oligonucleotide type. Set them from Register Item in the actions menu, which opens Edit Registration on a primer.
Ordering a Primer
Order, under those details, opens Request an order on Something new, filled in from the primer:
| Field | Filled with |
|---|---|
| Item name | The identifier and the primer's name, such as OL-0007 pUC19 forward primer 413 |
| Notes | The bases written 5' to 3' with their length, then the scale and the purification when the primer records them |
Choose the supplier and the quantity and record the request as you would any other. Work with Suppliers covers the rest of the dialog box and what happens to an order afterwards.
Once the request is recorded, Ordered appears beside the primer's name with the date, such as Ordered Oct 4. Click it for Order again, and the date moves to the latest request.
Every order requested this way also appears under the primer's Connections, in the actions menu at the right of its top bar. Click an order there to open it in Orders.
Ordering needs edit access to the project. A view-only member sees the date with no Order button.