Choosing a plasmid construction route by what the construct has to become: where golden gate assembly and the type iis restriction enzymes behind it beat overlap methods for multipart builds, what the types of restriction enzymes and a methylation sensitive restriction enzyme still settle that a modern assembly cannot, why dpn1 and dpni digestion is the step that decides whether a site-directed mutagenesis or site directed mutagenesis reaction gives clones or background, where a mutagenesis service, site saturation mutagenesis and saturation mutagenesis are cheaper bought than built, what dna ligase, t4 dna ligase, t4 ligase, t4 rna ligase 1 and t4 rna ligase 2 are each actually for, and when a plasmid cloning service, a dna cloning service, a gene cloning service, cloning service or cloning services work plus orf clones off the shelf is the honest answer

Choosing and running the build

1. Count the parts and the reuse. One insert into one vector is a job for any method. Four or more parts, or a series of constructs sharing a part library, is where a standardised assembly with defined junctions repays the setup. If you will build this family again, build the library once.
2. Check the sequence for the method's blind spots. Type two restriction assembly needs the chosen site absent from every part, and a single internal occurrence silently ruins the design. Overlap methods dislike repeats and strong secondary structure. Screen the sequence against the method before ordering anything.
3. Design mutagenesis around template removal. Amplification based mutagenesis produces a mixture of new product and methylated parental template, and the digestion that removes the parental plasmid is the whole game. Skipping or under running it produces colonies that sequence as the original, which is the most common wasted week in this area.
4. Match the ligase to the substrate. Standard double stranded ligation, single stranded ligation and adaptor joining are different enzymes with different buffers and different optimal temperatures. Using a general purpose enzyme on a specialist substrate produces a low yield that is usually blamed on the insert.
5. Sequence the whole construct, not the junctions. Junction sequencing was a reasonable economy when reads were expensive. Whole plasmid sequencing now costs little and catches backbone rearrangements, unintended point changes and the wrong parent entirely, all of which junction reads miss.
6. Decide build against buy honestly. A synthesised, sequence verified construct delivered ready to use frequently costs less than the labour of building it, particularly for difficult sequences or when a catalogue clone already exists. Price the bench time before defending the build.

Where each method earns its place

Restriction and ligation remains the clearest route for a single insert into a well characterised vector, and it is easy to teach and to troubleshoot. Overlap based assembly removes the site constraint and suits two or three parts with designed junctions. Type two site assembly comes into its own when the same parts recombine into many constructs, because the junctions become a standard rather than a design decision each time.

The cost of choosing wrongly is not the reaction, it is the colony screening. A method mismatched to the construct produces a low fraction of correct clones, and that is where the time goes.

Sequence verification is part of the build

A construct is not finished when a colony grows. It is finished when its full sequence is known and recorded against an identifier that the laboratory will still recognise in two years. That record should hold the sequence, the parent, the method, the date and the person, and it should live somewhere other than a laptop.

Groups that maintain this discipline rebuild their constructs rarely. Groups that do not rebuild them constantly, usually because nobody can prove what a tube contains.

What to ask a cloning service

What is delivered: plasmid at what quantity and purity, in which strain, with what sequence evidence, and whether the raw reads come with it. Who owns the construct and any intermediate. What happens if the sequence proves unbuildable, which for repeat rich or extreme composition sequences is a real outcome.

Also ask what happens next time. A service that keeps your part library and can rebuild variants quickly is worth more than a marginally cheaper one that starts from nothing each order.

Common questions

Which assembly method should a laboratory standardise on?

Whichever matches its most common job. Groups building multipart constructs repeatedly benefit from a standardised type two site assembly with a maintained part library; groups making occasional single insert constructs rarely need more than overlap assembly or ordering the construct.

Why do my mutagenesis reactions give the original sequence?

Almost always incomplete removal of the methylated parental template. Check the enzyme is active, extend the digestion and confirm that the input plasmid came from a strain that methylates, because template from a methylation deficient strain will not be cut at all.

Is whole plasmid sequencing worth it every time?

Yes, for anything that will be used more than once or shared. The cost is small against the cost of discovering a backbone rearrangement after six months of experiments built on the construct.

When does an off the shelf ORF clone beat a custom build?

Whenever the sequence, the tag position and the vector backbone you need already exist in a catalogue. The saving is not only the build but the sequence verification and the risk, and the main thing to check is the exact isoform and the licence attached to it.

Browse by service class

• Compare Gene synthesis on advertised prices