Cloning is a chain of small steps, and most failures trace back to one of them. Here is the whole chain, the reagent each step needs, and how to check you have the right clone before you build on it.
Range: Molecular biology reagents4 minute readUpdated
Most cloning problems are design problems. Before ordering primers, decide:
Amplify the insert with a high-fidelity, proofreading polymerase. Its 3′→5′ exonuclease removes misincorporated bases, so far fewer clones carry PCR errors than with standard Taq. Add what your method needs to the 5′ ends of the primers:
Check the product on a gel against a DNA ladder. If there is more than one band, gel-purify the right one; otherwise a PCR clean-up is enough.
| Method | How it works | Strengths | Watch for |
|---|---|---|---|
| Restriction-ligation | Cut insert and vector with the same enzymes; join with T4 DNA ligase | Directional with two enzymes; widely used | Internal sites in the insert |
| TA cloning | Taq-made A overhangs pair with T overhangs on the vector | Fast; no restriction sites needed | Not directional; needs A-tailing after proofreading PCR |
| Blunt-end | Blunt insert into a blunt-cut vector | Works with proofreading products | Low efficiency; not directional |
| Overlap assembly | Exonuclease, polymerase and ligase join fragments with matching overlaps | Scarless; several fragments at once | Overlap design; repetitive sequences |
For ligation, a vector-to-insert molar ratio of about 1:3 is a good start. Dephosphorylating a vector cut with a single enzyme stops it re-closing without an insert.
Only sequencing proves the insert is correct, in frame and free of point mutations. Universal primers bind standard vector sequences flanking the cloning site, so one primer works for any insert in that backbone.
| Primer | Binds |
|---|---|
| M13 forward and M13 reverse | Either side of the lacZα cloning site in many cloning vectors |
| T7 promoter | Upstream of the cloning site in T7-driven vectors |
| SP6 | In vectors carrying an SP6 promoter |
| Vector-specific reverse primers | Downstream sequences such as polyadenylation signals in mammalian vectors |
Once verified, make a glycerol stock of the colony and a larger plasmid prep for your experiments.
| Problem | Likely cause | Fix |
|---|---|---|
| No colonies | Low competent-cell efficiency, wrong antibiotic, failed ligation | Run a control plasmid transformation; check the antibiotic |
| Many colonies, none with insert | Vector re-closing without insert | Dephosphorylate the vector; gel-purify it after digestion |
| Insert present but mutated | PCR errors | Use a high-fidelity polymerase; reduce cycle number |
| Insert in the wrong orientation | Non-directional method | Use two different restriction enzymes or assembly |
The molecular biology range covers cloning systems, DNA ladders and polymerases for each step. To measure expression of the finished construct, see the qPCR master mix guide.
Design the construct, make the insert by high-fidelity PCR, join insert and vector, transform competent bacteria, screen colonies, and verify the clone by sequencing before growing a plasmid stock.
Primers that bind standard sequences in a vector rather than in your insert, such as M13, T7 and SP6 primers. Because they bind the backbone, the same primer can sequence any insert cloned into that vector.
TA cloning is fastest when you need to capture a Taq-amplified PCR product, but it is not directional. Restriction cloning with two enzymes is directional, which matters for expression constructs.
Proofreading polymerases correct misincorporated bases, so far fewer clones carry mutations. Standard Taq has no proofreading and is better kept for screening.
For research use only. Protocols here are general outlines: follow your kit manual and your institution’s safety rules. See our editorial policy.
Products for this workflow
Cloning systems, DNA molecular weight standards and DNA polymerases for cloning, PCR and gel analysis.
Molecular biology
A DNA ladder is only useful if its bands bracket your fragment on the gel you are running. This guide matches ladder ranges to agarose percentages and covers loading, staining and quantification.
4 minute read
DNA and RNA extraction
Blood brings heme, plants bring polysaccharides, stool brings humic acids and tough bacteria, viral samples bring almost no RNA. Here is what each sample needs from an extraction kit.
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Molecular biology
Read the gel first. No band, a smear, extra bands and a fuzzy band under 100 bp each point to a different change, and the positive control tells you whether to trust the mix.
3 minute read
Send the product names or catalogue numbers, quantities and delivery country. We'll confirm availability, lead time and the documents for molecular biology reagents.