Question 1

How is the melting temperature calculated?

Accepted Answer

From nearest-neighbour thermodynamics using the unified parameter set of SantaLucia (1998), with that paper's monovalent salt correction on entropy. Magnesium and dNTP enter as the monovalent equivalent of von Ahsen et al. (2001), and DMSO is applied as that paper's −0.75 °C per percent. Those authors report a standard error near 1.8 °C for perfectly matched duplexes under PCR conditions, so a predicted Tm is an estimate, not an exact annealing temperature.

Question 2

Does this tool check primer specificity against a genome?

Accepted Answer

No. Thermodynamic quality says nothing about whether a primer pair has other targets, and this tool performs no sequence database search. Run the candidates through a specificity tool such as Primer-BLAST before ordering. The tool states this limitation in its advisory section rather than leaving it implied.

Question 3

What does the primer ranking actually mean?

Accepted Answer

It counts how many of the sourced recommendation bands a candidate falls inside, weighting a pairing that reaches an extendable 3' end more heavily. A lower score means fewer deviations from documented criteria. It is a screening order, not a prediction that the PCR will work.

Question 4

Why are two different amplicon ranges shown?

Accepted Answer

Because published practice differs by application and the two windows do not overlap. A quantification assay and a cloning reaction have genuinely different constraints, so both ranges are shown with their own sources and the choice stays with you. The same applies to the 3'-end GC composition, where an unpublished lab convention points the opposite way from the mispriming concern.

Question 5

Why are the probe chemistries graded separately?

Accepted Answer

Because their constraints come from different mechanisms. A hydrolysis probe needs enough length to sit above the annealing temperature; an MGB conjugate reaches the same stability at roughly half that length, which Kutyavin et al. (2000) measured directly; a molecular beacon is a hairpin whose stem must hold the reporter and quencher together until the loop finds its target. Applying one chemistry's length band to another would be wrong, so the tool never does it.

Question 6

Does the probe Tm include the MGB or LNA modification?

Accepted Answer

No. Every Tm here uses unmodified-DNA nearest-neighbour parameters. A minor groove binder raises duplex stability above that estimate, so an MGB probe will run hotter in practice than the number displayed. For that reason the MGB probe's Tm gap over the primers, and its GC content, are shown but not graded against the unmodified-probe bands; check the Tm with your supplier's MGB-aware calculation.

Question 7

Why will the tool not generate molecular beacon stem arms?

Accepted Answer

A beacon stem has to be stable at the annealing temperature yet still openable by the target, and no citable rule set was located that fixes the arm sequence. Generating arms would mean inventing an unsourced heuristic and presenting it with the same weight as the cited rules. Instead the tool analyses a beacon you supply, states the stem length requirement, and leaves the arms to you.

Question 8

What does the probe-to-primer Tm check rely on?

Accepted Answer

The ordering follows from the assay mechanism: the probe must already be bound when extension reaches it, so its Tm sits above the primer Tm. The specific 5-10 °C band, however, is marked as evidence not yet verified, because no primary source was located for that number during this build. The tool reports the gap rather than borrowing a citation.

Question 9

Which plasmid features does the tool detect, and how reliable is each method?

Accepted Answer

In descending order of reliability: a GenBank FEATURES block is used exactly as submitted; affinity and epitope tags are found by translating open reading frames and matching standard peptides, which is codon-independent; short standard nucleotide motifs such as the T7 promoter, loxP and FRT are matched directly; and the multiple cloning site is computed from unique-cutter density. Every feature row shows which of these produced it, so an inference never looks like a record.

Question 10

Why does it not label the CMV promoter or the resistance cassette?

Accepted Answer

Those are long elements. Recognising them needs a reference sequence set and alignment, not a short signature, and guessing would produce a map that looks authoritative while being wrong. The tool lists what it did not look for as part of the result, so a missing feature is distinguishable from an absent one. Paste a GenBank record to pick up its own annotation for these.

Question 11

Are the restriction enzyme recognition sequences verified?

Accepted Answer

Not in this build. They follow standard REBASE and NEB notation, but outbound access to REBASE was unavailable in the build environment, so no automated cross-check ran. The provenance panel states this, and the table is published with its verified flag set to false. Confirm an enzyme against REBASE or your supplier before ordering oligos or setting up a digest.

Question 12

How are circular sequences handled?

Accepted Answer

Scanning runs across the origin, so a restriction site or an open reading frame that straddles position 1 is still found, and reported positions are normalised back into the 1 to length range. A feature whose span wraps the origin is kept as a wrap rather than being flattened into a full-length feature.

Question 13

Does the validation checklist mean my construct is correct?

Accepted Answer

No. The mechanical items resolve themselves; the ones that matter most stay marked for manual confirmation, because a correct map does not establish that the molecule in the tube is correct. Junctions and any PCR-derived region still need sequence confirmation.

Question 14

Is my sequence uploaded anywhere?

Accepted Answer

No. Parsing, thermodynamics, structure screening, ranking and plasmid analysis all run in your browser. Nothing is sent to a compute API and nothing is stored.

Primer, Probe & Plasmid Design

References

What the current settings may do

References

What the current settings may do