cDNA cloning produces a full-length transcript containing the 5′ and 3′ UTRs. It is usually inserted into a cloning vector and used as a PCR template, sequencing template, or for gene-function studies; subcloning is normally required before protein expression. ORF cloning includes only the coding region from the ATG start codon to the stop codon, inserts it into an expression vector such as pCMV, and includes a Kozak sequence for direct transfection and protein expression. Choose ORF cloning for protein expression and cDNA cloning when you need a gene template.

Subcloning transfers a fragment from an existing plasmid into a new vector after restriction digestion or PCR amplification. It is suitable for changing vectors, adding tags, or replacing promoters. PCR cloning directly amplifies the target fragment from cDNA, genomic DNA, or a plasmid template before cloning and is suitable for obtaining a gene from a sample for the first time. When the gene is already available in the In-Stock Gene Library, subcloning is faster and more economical.

Yes. Please provide the complete vector sequence, preferably sequence-verified, together with the plasmid map or information on antibiotic resistance and copy number, and specify the desired insertion site. If the sequence is incomplete, you may send the plasmid or a glycerol stock for sequence verification. Special restriction enzymes can be supplied by you or purchased on your behalf. If you do not have a suitable vector, you may choose from our library of more than 200 vectors at no additional cost.

We use homologous recombination, or seamless cloning, which does not depend on matching restriction sites in the vector and insert. A fragment can be directionally inserted at virtually any position in a vector in a single reaction, without multistep digestion and ligation. This approach provides a high positive-clone rate and is particularly suitable for incompatible restriction sites, large fragments, and multi-fragment assembly.

For standard sequences with normal GC content and no complex secondary structure, the success rate of a single-point mutation approaches 100%. Multi-site mutations or complex templates are assessed first, and the proposed strategy is discussed before work begins. All constructs are delivered after Sanger sequencing verification. One mutation unit is any nucleotide change within the region covered by one primer, approximately a 30 bp window. A single-base substitution, changing one codon across three consecutive bases, inserting a 10 bp tag, or deleting a 20 bp fragment each counts as one mutation site. Two distant sites that require two primer pairs count as two sites.

Standard cloning takes approximately 1–7 calendar days for fragments under 2 kb, 7–12 days for 2–3 kb, 12–15 days for 3–4.5 kb, and 15–20 days for 4.5–5 kb. A single-point mutation takes 1–10 days, while a double mutation takes 10–25 days depending on plasmid size. Multiple mutations are assessed according to the number and spacing of the sites. Difficult sequences or projects using specialized vectors or host strains may require additional time.

We recommend whole-plasmid sequencing to confirm the sequence before cloning or mutagenesis. Unknown mutations already present in the template can lead to incorrect cloning or mutagenesis results and are difficult to troubleshoot. We can provide whole-plasmid sequencing as part of a one-stop service.

Yes, although they are considered challenging projects. We assess GC content, repetitive sequences, secondary structure, and other factors before providing a customized strategy. For large fragments, gene synthesis is often recommended because it does not require a template and can include codon optimization. Alternatively, the fragment may be obtained from a large-insert vector such as a BAC or PAC through homologous recombination.