Tag: plasmid
Protocol: Double Digestion of Plasmid DNA with FastDigest EcoRV and HindIII Restriction Enzymes
A 100 – 200 ng plasmid DNA is sufficient to analyze by restriction digestion. Here we describe a procedure to double digest plasmid DNA with FastDigest EcoRV and HindIII restriction enzymes (from ThermoFisher Scientific). This procedure can be used to check the presence of a DNA fragment cloned in a vector. Double digestion will release the fragment of the appropriate size.
Protocol: Size Determination of Plasmid DNA Using Agarose Gel Electrophoresis
Plasmids are circular DNA. Their size can not be determined in their native state as the migration of circular DNA in agarose gel is quite different and can not be compared with the linear fragments of ladder DNA. To know the approximate size of plasmid, the first step is to linearize the plasmid using a restriction enzyme that generates strictly a single cut. This results in a linear DNA that can be run on agarose gel and its size can be compared with the ladder sequence.
Protocol – Growing Large Volume of Liquid Culture of Escherichia coli for Large-Scale Plasmid Isolation
Large-scale isolation of plasmid requires a large volume of E. coli culture. Large scale plasmid isolation procedures are termed, midiprep (25 – 50 ml starting culture volume) and maxiprep (100 – 500 ml starting culture volume). A starter culture is initially prepared by inoculating a colony in a small volume (2 – 10 ml) of culture medium. Large culture volume is prepared by diluting starter culture in a ratio of 1: 100 to 1: 1000 in the growth medium.
Protocol – Preparing Liquid Culture of E. coli for Plasmid Miniprep
Overview:
Small-scale plasmid isolation, called plasmid miniprep, requires a small amount of bacterial culture. Normally, a 2 – 5 ml culture provides sufficient cells that can be processed for plasmid isolation using the miniprep protocol. Culture can be prepared by inoculating 3 ml antibiotic-containing growth medium with a single colony and growing it overnight at 37C with shaking.
Here we have taken an example of preparing liquid culture from a colony of E. coli DH5α, transformed with the pEGFP plasmid. The pEGFP plasmid contains a kanamycin resistance gene, therefore, requires kanamycin for the selection of plasmid-containing bacteria. If your plasmid carries another antibiotic-resistant gene, add the respective antibiotic to the culture medium.