Plasmid construction
Dan Tudorica
Abstract
Via restriction enzyme digest and ligation
Steps
Design PCR primers such that they contain a BglII restriction site upstream of the gene, and SalI downstream of the gene. Design cut sites such that gene will still be in frame post ligation.
Acquire purified pmCherryC1 stock, and cleave using BglII and SalI using the standard NEB protocol. Simultaneously cleave PCR product
Purify both vector and insert using 1% agarose gel, and compare to uncut controls to make sure that restriction digest went to completion. Gel extract vector and insert.
Ligate with T4 ligase at room temperature overnight following standard NEB protocol.
Transform into XL10 Gold cells, plate on ampicillin selective medium, grow overnight at 37 C
Pick colonies, grow 5 mL overnight culture, and purify plasmid using a commercially available plasmid purification kit.
