The overall goal of this procedure is to generate safe, linear, covalently closed DNA minivectors, or DNA ministrings, using a simple, one-step production system. So this method can help answer key questions in the DNA vector and gene delivery field. In particular, how to efficiently produce safer and more efficacious gene delivery vectors called DNA ministrings.
The main advantage of this technique is that it is an efficient and simple, one-step in vivo approach for the production of DNA ministrings. To make media, after preparing 500 milliliters of LB, 600 milliliters of TB, and LB agar for plates, autoclave at 121 degrees Celsius for 30 minutes. After cooling, supplement each medium with ampicillin, at a final concentration of 100 micrograms per milliliter.
Store liquid media at four degrees Celsius. Then, pour 15 to 20 milliliters of the LB agar plus amp into sterile Petri dishes, and after cooling, store upside-down at four degrees Celsius. Streak W3NN PDMS cells on LB agar plus ampicilin and incubate the plate at 30 degrees Celsius overnight until single colonies can be observed.
Then, with a single colony from the streak plate, prepare an overnight culture of W3NN PDMS by inoculating five milliliters of LB plus ampicillin. Incubate the culture at 30 degrees Celsius and 230 to 250 RPM overnight. In a 125 milliliter Erlenmeyer flask, inoculate 10 milliliters of LB plus ampicillin with 100 microliters of the W3NN PDMS overnight culture to arrive at a 1 in 100 dilution.
Incubate the culture at 30 degrees Celsius and 250 RPM until it appears turbid to the naked eye and reaches mid-to late-log phase. Next, transfer 1 milliliter of culture to a clean cuvette. Then, using a UV-Vis spectrophotometer with a wavelength of 600 nanometers and the medium used to grow the cells, set the blank before measuring the light absorbance of the culture.
If the culture has not yet reached the appropriate OD, return it to the shaker and check once again every half hour. Once the culture has reached mid-log phase, transfer it into sterile 50 milliliter centrifuge tubes. Pellet the cells by spinning at 10, 000 times g for 10 minutes.
Decant the clear supernatant into an appropriate biohazard waste container, and use 100 microliters of LB plus amp to resuspend the pellet by pipetting up and down or by vortexing. To induce ministring production, add 20 milliliters of LB plus amp to a 125 milliliter Erlenmeyer flask and heat to 42 degrees Celsius. Transfer the resuspended pellet into the preheated medium and incubate at 42 degrees Celsius and 250 RPM past log phase, or no longer than one hour for 20 milliliters.
Reduce the temperature to 37 degrees Celsius, and leave the culture for an additional 90 minutes. Then, further reduce the temperature to 30 degrees Celsius, and leave the culture shaking at 200 rpm for two hours. To harvest the ministrings, transfer the culture into sterile 50 milliliters centrifuge tubes.
Then centrifuge at 10, 000 times g for 10 minutes, and inspect for a pellet. Finally, decant the supernatant and use a commercial extraction and endotoxin removal kit to extract the plasmid. After DNA extraction, residual precursor plasmid, LCC prokaryotic backbone, and the DNA ministring will be recovered and can be separated by agarose gel electrophoresis, as seen here.
Prior to ministring purification, a qualitative assessment of ministring production can be made at this stage by comparing band intensity of the ministring to the parent plasmid. This figure summarizes ministring production efficiencies likely to occur under various conditions while carrying out the protocol. Ministring production efficiency was determined based on DNA band intensity.
Once mastered, this technique can be fully completed in less than eight hours, if it is performed properly. While attempting this procedure, it's important to remember to adhere to the correct temperature shifts. Following this procedure, other methods like agarose gel electrophoresis or endonuclease digestion can be performed in order to isolate ministrings or check production efficiency.
This technique can greatly enhance gene delivery to eukaryotic cells. After watching this video, you should have a good understanding of how to maximize DNA ministring production using our production platform. Don't forget that, when working with ethidium bromide, UV illuminators can be extremely hazardous, and adequate laboratory safety gear should always be worn when performing this procedure.
