📘 Tutorial & Result Interpretation
🧬 1. Molecular cloning basics
Cloning means inserting a piece of DNA (the insert) into a carrier DNA
(the backbone or vector) to form a recombinant plasmid. Once inside a
bacterial or eukaryotic host, the plasmid replicates and can express the insert.
Every strategy has two conceptual halves:
- Opening the backbone — cutting it with a restriction enzyme, PCR-linearising it,
or excising a cassette.
- Joining the insert — via compatible overhangs (restriction, TA), overlapping homology
(Gibson), or type IIS overhangs (Golden Gate).
Key rule: Orientation and reading frame matter. A correct insert in the wrong direction
or the wrong frame will not express the desired protein.
🚀 2. General workflow
- Obtain the backbone — from Addgene, NCBI, or your lab's glycerol stock. Note whether it is circular.
- Obtain the insert — PCR-amplify, order as a gBlock, or extract from another plasmid.
- Choose a cloning method — restriction (classic), Gibson (scarless), Golden Gate (multi-part), TA (fast, non-directional).
- Run this tool — paste both sequences, pick the method, inspect the primer suggestions and warnings.
- Order primers or the synthetic insert, then perform the assembly and transform into E. coli.
- Validate — colony PCR, restriction digest, and finally Sanger sequencing of the junctions.
Tip: Always sequence-verify both junctions of your final plasmid. Even a correctly-designed
assembly can carry a PCR-introduced mutation.
✂️ 3. Restriction cloning — reading the output
What the tool does
It scans the backbone for the two chosen enzymes, scans the insert, and — if the insert is
clean — builds the exact primers you need (recognition site + annealing region), reports
the overhang type at each junction, and warns about problems.
The "site count" cards
| Value | Meaning | Action |
|---|
| 1 site in backbone | Perfect — a clean cut. | Proceed. |
| 0 sites in backbone | The enzyme does not cut this vector. | Pick another enzyme. |
| >1 site in backbone | Multiple cuts — the vector will be fragmented. | Use a partial digest, gel-purify the correct fragment, or switch enzymes. |
| 0 sites in insert | The enzyme does not cut inside your insert. | Proceed — the insert is protected. |
| >0 sites in insert | The enzyme cuts the insert internally. | Remove the site by silent mutation, or pick a different enzyme. |
Primer layout
- Tail (amber) — the recognition site, added 5′ of the insert-specific region.
It is not counted toward Tm.
- Annealing region (blue) — matches the insert end; typically 20–24 nt with 40–60 % GC.
Overhang table
| Overhang | What it means | Ligation efficiency |
|---|
| 5′ X nt | 5′ protruding end (EcoRI, BamHI, HindIII…). | Highest — standard. |
| 3′ X nt | 3′ protruding end (KpnI, SacI, PstI…). | Good — but check compatibility. |
| blunt | No single-stranded overhang (SmaI, PmeI…). | Lower — add 5′ phosphate or use a blunt-end kit. |
Reading the junction rows
Each junction shows the left arm (the enzyme site or insert end) and the right arm
(the insert start or the reverse-complement of the second enzyme site). If the two ends of
the insert carry different overhangs, cloning is directional — the insert can only
go in one way, which is what you want.
Same enzyme at both ends? The insert can ligate in either orientation. Either use two
different enzymes, or screen many colonies by PCR to find a correctly oriented clone.
🔗 4. Gibson assembly — reading the output
What the tool does
Gibson is scarless — no restriction sites are left behind. The backbone is linearised by
PCR (you specify the coordinates to keep), and every fragment carries 20–40 bp of homology to
its neighbour. The tool computes those homology arms and produces primer pairs.
Inputs you must provide
- Linearise backbone start / end — the region to keep. The rest is removed and replaced by the insert.
- Overlap length — typically 25 bp; extend to 40 bp for AT-rich regions.
Reading the primer table
- Name —
Gib_F1 is the forward primer of fragment 1, Gib_R1 its reverse partner, etc.
- Full primer — the amber region is the homology arm, the blue region is the 3′ annealing region.
- Notes — says which fragment the homology arm matches.
Rule of thumb: design 2 fragments for a simple insert; 3–5 fragments for a pathway.
Purify each fragment by column before assembly. Incubate at 50 °C for 15–60 min.
🏛️ 5. Golden Gate assembly — reading the output
What the tool does
Golden Gate uses type IIS enzymes (BsaI, BsmBI, BbsI…) which cut outside their
recognition site, so the site itself is removed from the final product. Each fragment carries
the recognition site followed by a one-base spacer and a 4-bp overhang. Overhangs must be
unique and non-palindromic.
What the tool checks
- Internal sites in the backbone and inserts — these must be "domesticated" (silently
mutated) or the assembly will fail.
- Overhang uniqueness — a repeated overhang means wrong fragments can ligate together.
- Self-ligation — the 4-bp overhang must not recreate the recognition site after ligation.
Reading the output
- Primer tail — recognition site + 1-nt spacer + 4-nt overhang. This is what you order.
- Junctions — the chosen 5′ and 3′ overhangs. Confirm they match your design.
- Warnings — see above; address all of them before ordering.
Choose overhangs carefully. A good rule: avoid overhangs that are palindromic, that
differ by only one base from another overhang in the same reaction, or that end in a run of
Ts.
⚡ 6. TA / blunt cloning — reading the output
Fast and simple, but non-directional. The vector carries a 3′ T overhang; the insert
(produced by Taq) carries a 3′ A overhang. They anneal and ligate.
| Observation | Interpretation | Action |
|---|
| Backbone/insert < 20 bp | Suspiciously short fragment. | Re-check the sequence you pasted. |
| Both A and T detected near the ends | Non-directional ligation expected. | Screen colonies by PCR or sequencing for orientation. |
When to use TA: quick subcloning, PCR product storage, or a template for sequencing.
For expression constructs, prefer restriction or Gibson.
🗺️ 7. Restriction mapping — columns explained
| Column | Meaning |
|---|
| Position | 1-based coordinate of the first base of the recognition site on the + strand. |
| Enzyme | The restriction enzyme that recognises the site. |
| Site | The exact DNA sequence that was matched. For − strand hits this is the reverse complement of the + strand site. |
| Strand | Which strand carries the recognition sequence. |
| Overhang | Type and length of the cohesive end produced by cleavage. |
| Cut position | Two numbers — the cut coordinate on the top strand and on the bottom strand. Equal = blunt; unequal = overhang. |
| Wrap-around | For circular plasmids only: the site spans the origin. Very useful for accurate map ordering. |
On a circular plasmid, tools often miss sites that cross the origin — the wrap-around
column tells you which hits required circular extension, so you can trust the map.
🧾 8. ORF finding — columns explained
| Column | Meaning | Typical values |
|---|
| Start / End | Coordinates of the ATG and the last base before the stop codon. | 1-based |
| Length (bp) | Distance from ATG to the stop codon, inclusive. | ≥ 300 bp for a real protein |
| Amino acids | Length ÷ 3 − 1 (excluding stop). | ≥ 100 aa is convincing |
| Frame | Which of the 3 reading frames contains the ORF. | 1, 2 or 3 |
| Strand | + (same as input) or − (reverse complement). | Both are valid |
| Stop | The stop codon that ends the ORF (TAA, TAG, TGA). | — |
| Peptide preview | First ~13 amino acids translated in the standard genetic code. | — |
ORF ≠ gene. A long ORF on a bacterial plasmid is often a selection marker or a
fragment of one. Cross-reference with the expected protein.
📈 9. GC content profile
The sliding-window plot shows how G+C content varies along the backbone. Regions that are
unusually AT-rich or GC-rich tell you something about the biology of the sequence.
| Pattern | Likely interpretation |
|---|
| Flat around 50 % | Typical coding or mixed sequence. |
| Local AT-rich dip (< 35 %) | Promoter / regulatory region, or an origin of replication. |
| Local GC-rich peak (> 65 %) | rRNA / structural RNA gene, or a GC-rich coding region — may be hard to amplify. |
| Very sharp spike | Often a tandem repeat, a homopolymer, or an artefact in the input. |
Rule of thumb: a window of 100 bp is a good default; use 500 bp for whole-plasmid
trends and 20–50 bp for pinpointing problematic regions.
⚠️ 10. Common pitfalls
Reading frame. When cloning into an expression vector, the insert must be in-frame with
the promoter's ATG. Use the ORF finder to check; the tool does not check frame automatically
for you.
Dam / Dcm methylation. Some enzymes (ClaI, XbaI in certain contexts) are blocked by
Dam methylation in E. coli. If your digest fails, prepare the DNA from a dam⁻ strain.
Restriction site in the insert. Always check the "insert sites" card before ordering —
a single internal site ruins the entire strategy.
Golden Gate overhang collisions. With 4-bp overhangs and more than four fragments, the
probability of a palindromic or duplicated overhang rises sharply. Verify every overhang pair.
Best practice. Sequence both junctions of every final construct. A single-base error
introduced by PCR or by synthesis can silently inactivate your protein.
Pre-order checklist
- ✔ Each enzyme cuts the backbone exactly once.
- ✔ No internal sites inside the insert.
- ✔ Overhangs are compatible and give the correct orientation.
- ✔ Primers have 40–60 % GC and Tm within 3 °C of each other.
- ✔ Golden Gate overhangs unique and non-palindromic.
- ✔ Reading frame checked if expressing a protein.
- ✔ Final plasmid sequence verified by Sanger before any downstream work.