유전자 기반 연구
유전자 기반 연구

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/µg

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.