LakePharma provides plasmid design and construction services that include subcloning a gene of interest into a vector of choice, as well as site-directed mutagenesis to introduce point mutations, small insertions, or deletions. Gateway cloning is also available, as is custom cloning in which a peptide tag or additional sequence is encoded at the N or C terminus.
For more information or to request a custom cloning project, please email email@example.com
1. The gene of interest (provided in a plasmid or as a PCR product flanked by appropriate restriction enzyme sites) is digested with restriction enzymes and subcloned into the vector of interest, which contains compatible sites.
2. Plasmids containing the insert are confirmed by restriction digest. The inserts can be sequenced for an additional cost.
1. The gene of interest is amplified with primers containing appropriate restriction enzyme sites.
2. The PCR product is digested with restriction enzymes and subcloned into the plasmid of interest containing compatible sites.
3. Plasmids containing insert are confirmed by sequencing (for an additional cost, see below)
Please note that DNA sequencing of the cloned PCR product is not included as a standard part of this service but is provided by LakePharma at an additional cost (see DNA sequencing and analysis link below). Sequencing the cloned PCR product is necessary to confirm that no mutations were introduced during PCR amplification.
1. Primers are designed and used to perform the mutagenesis reactions.
2. Sequencing of the mutated portion of the gene is included to confirm that the desired change was incorporated.
Please note that there will be an additional charge to sequence the remainder of the insert to ensure that no additional mutations were introduced during the mutagenesis reaction.
1. A gateway LR recombination reaction is performed between the attL entry clone and the Gateway destination vector. (If a gateway entry clone is needed, this can be generated as a separate PCR cloning project.)
2. Clones with the insert are verified by restriction enzyme digest or sequencing.
Note: midiprep DNA or higher is needed for entry and destination vectors. If miniprep DNA is provided by client, Midiprep scale-up service (cat# 21602) will be included.
1. Additional sequences, such as a peptide tag or protease cleavage site, can be added using PCR.
2. Correct clones are identified by sequence analysis.
1. DNA template (PCR product or gene within a plasmid)
2. 1-2 ug vector (LakePharma offers several vector options--please inquire)
3. Appropriate sequence information
4. For gateway cloning, midiprep DNA or higher is needed for entry and destination vectors. If miniprep DNA is provided by client, Midiprep scale-up service (cat# 21602) will be included.
Please complete and include a DNA Studio Order form with any vectors or plasmids you send to LakePharma.
1. Recombinant plasmid from a miniprep
2. Certificate of analysis