Plasmid Linearization - In Vitro Grade

Plasmid Linearization Process

Following plasmid purification (midiprep, maxiprep, or gigaprep scale), the plasmid is digested with a restriction enzyme that has a single recognition site located outside the expression cassette. This standard linearization produces a fragment equal in size to the full plasmid.

Alternatively, the plasmid can be digested with two restriction enzymes to excise only the expression cassette, removing the surrounding vector backbone. In both cases, the linearized DNA is cleaned and eluted in Tris-EDTA buffer.

Applications of Linearized Plasmids

Plasmid linearization is essential for applications where circular DNA is incompatible or less efficient:

• In Vitro Transcription (IVT): Linear templates are required for mRNA synthesis using T7, SP6, or T3 RNA polymerases, ensuring transcription terminates cleanly.
• Cell-Free Protein Synthesis (CFPS): Linear DNA is often preferred in TX-TL systems due to its compatibility with open reaction conditions and reduced metabolic burden.
• Gene Circuit Prototyping: In synthetic biology, linear DNA supports rapid design–build–test cycles where constructs are assembled, expressed, and degraded without cloning.
• IVT-based Genome Editing Reagents: Used to generate synthetic mRNAs for Cas9, base editors, or guide RNAs in gene editing workflows.

Quality Control

We provide high-quality linearized plasmids at all scales, from research to preclinical use. Every preparation undergoes a four-point QC process to ensure it meets the highest standards:

1. UV absorbance at 230/260/280 nm for concentration and purity
2. NGS-based identity confirmation of the plasmid sequence
3. Endotoxin testing to verify < 0.1 EU/µg (endotoxin-free)
4. Capillary electrophoresis to confirm >95% linearization

Linearized DNA is delivered in Tris-EDTA (10 mM Tris, 1 mM EDTA, pH 8.0) at a concentration of 500 ng/µL. Alternative formats, such as nuclease-free water or lyophilized DNA, are available upon request.

Limitations

• Residual circular DNA: Even after restriction digestion, linearized plasmid solutions may contain up to 5% circular plasmid. This is not an issue for IVT or CFPS, but not suitable for cloning or applications requiring pure linear fragments.
• Dual-enzyme digests: When using two restriction enzymes to isolate the expression cassette, the resulting DNA mixture may contain multiple fragments and residual circular plasmid. The standard service does not include fragment isolation via gel or column separation.
• Not suitable for library generation: Because of residual circular DNA, plasmid libraries or recombination workflows should use our Plasmid Fragment – Cloning Grade service instead.
• Available only for GenoCAD plasmids: To ensure quality and process integrity, this service is only available for plasmids synthesized and purified by GenoCAD. We do not accept external plasmid samples for linearization.