유전자 기반 연구
유전자 기반 연구

Growth Optimization for Plasmid Purifications

Is the purification process compatible with TB and other rich media?

Yes, the PhyNexus plasmid purification columns are compatible with TB and all other rich media. Because the AutoPlasmid MMG was has a high capacity buffer system, we recommend using rich media to obtain the highest amount of plasmid DNA.

Is the plasmid purification process compatible with LB media?

Yes, the PhyNexus plasmid purification columns are compatible with LB and other simple media. However, the amount of plasmid DNA achieved using these media types is typically lower.

What size plasmids are compatible for purification on the AutoPlasmid MMG?

The AutoPlasmid MMG and kits are compatible with all plasmid types. It is recommended that low copy plasmids be cloned to medium and high copy vectors to obtain higher yields when possible.

What host cells are compatible for purification on the AutoPlasmid MMG?

Competent cells used for transformation of plasmid and ligated material can be generated in house or purchased. Sources and types of competent cells include XL1-Blue (Agilent) and DH5α (Invitrogen) for transformation efficiency of 1 x 106 transformations per microgram of DNA. These sources and laboratory preparations of competent cells are recommended when DNA is abundant (1-10 ng per reaction).

When DNA is less abundant, commercial sources of ultracompetent cells are recommended. These cells feature genes or genetic mutations that increase competence, decreases loss of DNA, resistance to and/or decreased endonuclease activity. Commercial strains of ultracompetent cells include One Shot TOP10 cells from Invitrogen and XL2-Blue from Stratagene which report transformation efficiency of 5 x 109 transformations per microgram of DNA.

Ultracompetent cells are also recommended for transformation of large clones. In addition, if processing times are limited, Mach 1 cells may be used for their increased growth efficiency. Double times are reduced from 74 minutes to 50 minutes when compared with DH5α. Plasmid preps can be carried out in as little as 4 hours after inoculation. However the yield is dictated by the downstream requirements.

What culture volumes should I grow?

For best results, we recommend using a culture volume based on cell mass.  A denser culture will result in a larger cell pellet wet weight, while a less dense culture will result in a smaller cell pellet wet weight.  A low cell mass will result in lower than expected DNA yields regardless of the culture volume used.  Therefore, it is always better to use denser cultures to achieve high DNA yields. Typically, for a miniprep and midiprep purifications, a culture volume of  1.4 mL and 5 mL is sufficient. For maxi, mega and gigapreps, PhyNexus recommends cultures volumes of 400 mL, 1.5 L, and 3 L, respectively. If you need more plasmid DNA, you can always use more culture.

How long should I grow the culture?

Cultures should be grown to obtain largepellet wet weights. Generally, cultures should be grown for at least 16 hours.

At what RPM should I shake the culture?

The incubator setting is dependent upon the amplitude of the shaker, type of flask being used, and the culture volume to flask ration. For a New Brunswick I26 Incubator/Shaker with an orbit of 2.5 cm 350 rpm is used to grow cultures.

Is there a recommended culture flask?

It is recommended to use baffled flasks such as the Thomson Ultra Yield.

What should be used to seal the culture flasks?

AirOtop Enhanced Seals from Thomson have a very high rate of air exchange.

How to I purify more plasmids?

The flexibility and robustness of the AutoPlasmid MMG and kits mean that ideal mass of plasmid can be achieved. Generally, if more plasmid is desired, then the user can grow larger cultures.