Cloning cDNA Into Vector

This section provides general guidelines and protocols for insert design and preparation, restriction digestion of cDNA vector, and cloning of your cDNA fragment.

General Guidelines for cDNA Insert Preparation

Full length cDNA fragments can either be recloned from another plasmid or amplified by PCR. PCR-based cloning is the most convenient way for full-length cDNA cloning in Cellecta’s cDNA and InDOXible lentiviral vectors. The cDNA vectors do not contain ATG initiation codons, so this translation initiation sequence must be included in the insert to be cloned if it does not already have one. We also recommend including a Kozak sequence (i.e. GCCACC) before the ATG codon for optimal translation.

For amplification of the target cDNA fragment from another plasmid, design a 5’-primer (containing both Kozak sequence and ATG codon) and 3’-primer with unique restriction sites present in the multiple cloning site (MCS) of the cDNA vector but absent from either strand of the cDNA sequence. Amplify the cDNA fragment by high fidelity long-distance PCR using about 200 ng of plasmid template DNA and a minimum number of cycles (usually 12-15 cycles), purify, digest the amplified product with end-specific restriction enzyme(s) and purify the digested PCR product in a 1.2% agarose gel (low-melting) to prevent contamination with the original plasmid used for amplification.