Enzymes & cloning

Cloning failure diagnosis (ranked causes from design + observation)

Work out why a cloning experiment failed: no colonies, every clone empty vector, or no PCR band. Takes your design (method, parts, enzymes, primers, host methylation state) plus what you actually observed (colony counts on the plate and on each control, screening tally, band sizes, whether the ladder ran) and returns causes ranked by evidence — each with the deterministic fact from the design or the observation that implicates it, the cheapest observation that would separate it from the next candidate, and the next experiment. Causes the observations eliminate are reported as eliminated, naming the observation that did it; causes the design makes impossible are not listed. No probability is computed anywhere — the ordering is of evidence, not of likelihood, and ranking.evidenceBased says so when the inputs separate nothing.

post/cloning_diagnose

Request

  • Base URL: https://seqbench.com/api/v1
  • URL: https://seqbench.com/api/v1/cloning_diagnose
  • Auth: none required

Request body

symptom'no_colonies' | 'wrong_clones' | 'no_pcr_band' required

The complaint. "no_colonies" = the plate is blank; "wrong_clones" = colonies grew but the clones are empty vector / rearranged / the original template; "no_pcr_band" = the gel lane is empty or the band is the wrong size.

method'goldengate' | 'gibson' | 'restriction_ligation' | 'pcr' required

How the construct was made. Decides which cause set applies and which design checks can run.

partsstring[]

Parts in assembly order (Golden Gate / Gibson), or [vector, insert] for a restriction ligation. Raw or FASTA. Up to 8; 300,000 bp total across parts and template.

partNamesstring[]

Labels for the parts, in the same order ("backbone", "promoter", "CDS"). Used in every fact that names a part.

enzymesstring[]

The Type IIS enzyme for Golden Gate (BsaI, BbsI, Esp3I (BsmBI)), or the one or two restriction enzymes the insert and vector were cut with. An unrecognized name is rejected rather than skipped: every methylation and site-geometry verdict here reads "no site found" when the enzyme is missing, so a typo would come back as a clean bill of health.

overlapLennumber

Gibson only — the homology arm length the assembly was designed with.

templatestring

PCR template, raw or FASTA.

templateCircularboolean

Treat the template as a plasmid, so a product across the origin is found.

primerForwardstring

Forward primer, up to 200 nt.

primerReversestring

Reverse primer, up to 200 nt.

annealingTempCnumber

Annealing temperature actually used, °C. Compared against each primer's own nearest-neighbor Tm.

agarosePercentnumber

Agarose percentage of the gel. Snapped to the nearest tabulated value; decides the resolving window a band is placed against.

hostMethylation'dam_dcm_plus' | 'dam_dcm_minus' | 'unknown'

Dam/Dcm state of the DNA that was CUT — a property of the strain the plasmid was prepared from, not the one it was transformed into. Standard cloning strains are dam+/dcm+. "dam_dcm_minus" eliminates every methylation-blocking cause; "unknown" leaves the sequence overlap stated as a fact and the methylation as an open question.

phosphataseTreatedboolean

Was the cut vector dephosphorylated (rSAP/CIP) before ligation?

insertSource'plasmid_pcr' | 'genomic_pcr' | 'synthesis' | 'digest' | 'unknown'

Where the insert came from. Only "plasmid_pcr" changes a verdict: a plasmid template replicates and selects on its own, so it can give colonies with no ligation at all.

vectorMarker'amp' | 'ampicillin' | 'bla' | 'cam' | 'carb' | 'carbenicillin' | 'chlor' | 'chloramphenicol' | 'gent' | 'gentamicin' | 'hyg' | 'hygromycin' | 'kan' | 'kanamycin' | 'neo' | 'spec' | 'spectinomycin' | 'strep' | 'tet' | 'tetracycline' | 'zeo' | 'zeocin'

Selection marker on the vector. Compared against the plate's antibiotic by identity, not by guessing at free text.

plateAntibiotic'amp' | 'ampicillin' | 'bla' | 'cam' | 'carb' | 'carbenicillin' | 'chlor' | 'chloramphenicol' | 'gent' | 'gentamicin' | 'hyg' | 'hygromycin' | 'kan' | 'kanamycin' | 'neo' | 'spec' | 'spectinomycin' | 'strep' | 'tet' | 'tetracycline' | 'zeo' | 'zeocin'

Antibiotic on the plate. amp and carbenicillin count as the same selection (both select bla).

coloniesnumber

Colonies on the experimental plate. Omitting this is different from giving 0.

noDnaControlColoniesnumber

Colonies from the same cells and plate with NO DNA added. Non-zero means the plate is not selecting.

positiveControlColoniesnumber

Colonies from an intact, uncut plasmid into the same cell aliquot. Non-zero eliminates "the cells never took up DNA".

positiveControlSameMarkerboolean

Did that control plasmid carry the SAME selection marker? If not, it eliminates the cells but says nothing about the plate — the tool reports the difference.

noInsertControlColoniesnumber

Colonies from the identical reaction with the insert (or one part) left out.

uncutVectorControlColoniesnumber

Colonies from the digested but UNLIGATED vector. Measures intact vector that survived the digest, with no help from the ligase.

transformationEfficiencyCfuPerUgnumber

cfu/µg from a control transformation, where it was measured.

gelBandsObservedBpnumber[]

Band sizes read off the lane, bp. An empty array ("I looked and there was nothing") is not the same as omitting the field.

ladderRanboolean

Was the ladder itself visible in the same gel? A true here eliminates the whole stain-and-imaging branch in one observation.

Response

Tool ran successfully.

okunknown required
toolstring required

The tool slug that ran.

resultobject required

Tool-specific output object. Its shape depends on the tool — call GET /{tool} or see the docs for each tool's fields.

Example response

{
  "provenance": {
    "apiVersion": "1.1.0"
  }
}

Changes

No recorded changes to this operation across all 1 revision of this API.