Automation and Large Scale Plasmid Purification Kits

What are the expected yields from a Mini, Midi, Maxi, Mega or Giga prep?

The number of bacterial cells and the amount of plasmid DNA per cell can vary greatly between cultures based on many factors (strain of E. coli, plasmid copy number, media type, growth conditions). The maximum yields from a Mini, Midi, Maxi, Mega and Giga prep are 20 µg, 50 µg, 1.5 mg, 5 mg, and 10 mg, respectively. The typical yields from a Mini, Midi, Maxi, Mega and Giga prep are 10 µg, 25 µg, 750 µg, 3.5 mg, and 7 mg, respectively.

What are the advantages of the PhyNexus Mini, Midi, Maxi, Mega, and Giga prep kits?

PhyNexus plasmid purification kits offer full automation that is reliable and requires minimal maintenance. The kits are robust and compatible with a wide range of starting culture volumes.The purified plasmid DNA is transfection quality and can be eluted at high concentrations, eliminating the need for an additional ethanol precipitation step

What are the capabilities of the AutoPlasmid MEA?

The AutoPlasmid MEA is capable of purifying plasmid DNA in the mini or midi scale. The throughput is 48 or 36 samples per run, respectively. The instrument is also capable of running all of PhyNexus’ protein and chromatography PhyTip columns.

How is the AutoPlasmid MMG different from other technologies?

The AutoPlasmid MMG was designed to handle a wide range of starting samples.. Reliability is one of the most important qualities of laboratory equipment, and the AutoPlasmid MMG was built to be robust.  Combining a simple set-up with broad sample input requirements gives the AutoPlasmid MMG a distinct advantage over conventional manual purification kits.

What other consumables or accessories are needed besides the Maxi, Mega and Giga prep kit?

Required:

  • 20 mL transfer pipette tips (P/N DPM 00-10-00)
  • Sample prep conical tubes, pyrogen free, sterile conical tubes (P/N DPM 00-020-00)
  • 200 proof ethanol
  • Deionized water

Recommended:

  • AutoPlasmid MMG post-processing kit (P/N DPM 00-30-00)

 

Does the kit need to be refrigerated?

No the kit does not have to be refrigerated. The kit comes with RNase A and it is recommended to store that at -20 °C.

What is the expiration date on the kit?

The kit is guaranteed for 1 year after manufacturing date.

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.

Plasmid DNA Purification and Recovery Process

Why is it recommended to grow larger cultures than what is typically needed for a manual prep?

Automation of a process is not simply automating a manual process. Instead, a whole new technology must be developed to find solutions for manual steps that are not feasible to automate. It is recommended to input an excess amount of plasmid in order to guarantee the desired yield is achieved. The benefit of reproducible walk-away automation outweighs the cost of growing extra E. coli culture. To get more, grow more.

What form is the starting sample, the overnight culture, or cell pellet?

The AutoPlasmid MMG procedure begins with a cell pellet generated by centrifugation of an overnight culture volume.

Does the purification process use anion exchange?

No, the plasmid purification processes uses enhanced silica chemistry. This results in transfection grade plasmid DNA at usable concentrations without the need for ethanol precipitation.

Why is elution done using gravity?

Plasmid release from PhyTip columns requires gravity elution for high performance and high concentration. The elution utilizes the traditional chromatography principle of multi-equilibrium for elution of the plasmid DNA, which has a slow off-rate.

How many samples can I set up and walk away?

For mini and midiprep, it is possible to setup 48 and 36 samples, respectively. For maxi, mega, and gigaprep, it is possible to setup 4, 4, and 2 samples, respectively, for walk-away purification.

How many Maxi, Mega or Giga preps can I process in a day?

For Maxi, Mega and Giga preps, it is possible to purify 12, 8, and 4 samples in a day, respectively.

Can I run the AutoPlasmid MMG overnight?

Yes, it is possible to setup the MMG and complete the run unsupervised.

How can I increase my  yields?

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, the user can grow larger cultures.

What are the expected yields?

The maximum yields from a Mini, Midi, Maxi, Mega and Giga prep are 20 µg, 50 µg, 1.5 mg, 5 mg, and 10 mg, respectively. The typical yields from a Mini, Midi, Maxi, Mega and Giga prep are 10 µg, 25 µg, 750 µg, 3.5 mg, and 7 mg, respectively. What are the expected concentrations from a purification?

The concentration of plasmid DNA is dependent on the elution volume used during the process. For miniprep and miniprep, conecnetrations can reach >100 ng/µL. For Maxi, Mega, and Gigapreps, concentrations can reach 1mg/mL

What are the A260/A280 and A230/A260 ratios?

The expected A260/A280 and A230/A260 ratios are 1.8-1.9 and 2.0-2.1, respectively.

What is the resulting concentration of endotoxin in the purified sample?

Endotoxin levels are less than 10 EU/mg

Are the plasmids transfection grade?

Yes, plasmids purified by PhyTip columns are transfection grade

Is it necessary to perform ethanol precipitation at the end?

No, ethanol is not necessary. Endotoxin levels are within transfection range and concentrations are high enough for downstream use.

Are the purified samples free of ethanol?

Yes, samples are free of ethanol and measured to be less than 3%.

Are the purified plasmids sterile?

Yes, samples are sterile. However, the AutoPlasmid MEA and MMG are open instruments. It is recommended to process the samples through a post-purification kit which consists of a 0.2 micron syringe filter.