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Developed to Overcome the Limitations of Current Methods
Researchers have been performing plasmid minipreps for years—but they could be using an easier, faster, safer method and getting better results. Today, the majority of the plasmid miniprep methods in use require a silica membrane for DNA binding and purification. While generally effective, this technology suffers from a number of limitations, including the possible introduction of contaminants into downstream applications, compromising your results (Table 1). Other methods employ anion exchange to purify plasmid DNA. This method is effective but is expensive, slow, and requires a precipitation step during which you may lose sample. But, there is a better way—ChargeSwitch® pH-dependent technology. Now you can purify plasmids without introducing inhibitors such as ethanol and chaotropic salts and obtain a cleaner purified plasmid for optimal downstream performance in PCR, sequencing, and transformation experiments.
Table 1. ChargeSwitch® overcomes the limitations of traditional plasmid purification.
|Limitation||Implication||How This Affects You|
|Ethanol and guanidinium HCl are required to facilitate binding and removal of contaminants||Both reagents are difficult to remove from sample once introduced||Additional steps such as centrifugation or "smack and dab" methods are required, but typically not 100% effective|
|Both reagents are known to inhibit enzymatic activity in low concentrations||Reduced enzymatic activity and overall poorer enzymatic performance in common applications such as PCR, restriction digestion, and cloning|
|Both reagents are hazardous or require proper disposal in common applications|
|Binding mechanism is nonspecific adherence; ion exchange is the preferred format for quality performance||The ability to selectively purify the plasmid DNA is limited||Reduced purity and performance in common applications|
|Delicate glass-fiber membranes||The shear forces involved in centrifugation can result in release of small glass-fiber particulates||Optical density shifts affects quantitation
Fibers can damage microcapillary instrumentation
|Plasmid integrity is compromised||Supercoiled plasmid forms are generally preferred for optimal performance|
The ChargeSwitch® protocol retains the ease and familiarity of common silica spin-column protocols with binding, washing, and elution steps (Figure 1, Table 2), making adapting to the ChargeSwitch® method effortless.
Just Add Your Sample
The ChargeSwitch®-Pro Plasmid Miniprep Kit provides all reagents and components required to process your sample. There is no need to add ethanol to any buffers or search for additional collection tubes. All you need to supply is your E. coli culture.
Figure 1. The easy-to-follow ChargeSwitch®-Pro Plasmid Miniprep Kit protocol.
Table 2. ChargeSwitch® Pro Plasmid Miniprep Kit specifications.
|Culture Volume||1–5 mL (LB or TB)|
|Purity (A260/A280)||1.7 – 2.0|
|Yield||Up to 20 ug|
|Plasmid Size||Up to 20 kb|
|Plasmid Copy Number||High or low copy|
|Processing||Centrifugation or vacuum|
Since ChargeSwitch® Technology does not require the use of ethanol, chaotropic salts, or organic solvents for nucleic acid purification, the resulting DNA is free of potentially inhibitory compounds, improving DNA integrity for better downstream PCR performance (Figure 2). Expect to see improved cloning due to higher transformation efficiencies (Figure 3), faster restriction enzyme digestions (Figure 4), sequencing reads in excess of 800 base pairs with Phred scores >20, and a higher degree of supercoiling (Figure 5).
Figure 2. ChargeSwitch®-Pro reagents do not contain inhibitory components that may interfere in downstream PCR reactions. The inhibitory reagents used in most plasmid purification techniques are difficult to detect when measuring DNA quality on gels or by UV spectrophotometry. They can, however, inhibit PCR. The action gene was amplified by PCR from human placental DNA, in a 50-µL reaction spiked with varying volumes of ethanol, isopropanol (IPA), competitor wash buffers, guanidinium isothiocyanate (GTC) and ChargeSwitch® wash buffer. Ten microliters of amplified product run on a 1% agarose gel and stained with ethidium bromide.
Figure 3. Higher transformation efficiencies with plasmid purified using the ChargeSwitch®-Pro Kit. The ChargeSwitch®-Pro Plasmid Miniprep Kit and a competitor silica prep kit (Q) were used to purify pUC19 plasmid DNA from 5 mL of LB culture. One Shot® TOP10 Chemically Competent E. Coli was transformed using 10 pg of the purified pUC19 plasmid DNA, or OneShot® TOP10 Electrocomp™ E. Coli was transformed with 5 pg of the purified pUC19 plasmid DNA. pUC19 plasmid DNA supplied with the competent cells was used as a control. Error bars represent 1 SE (n=12).
Figure 4. Improved restriction enzyme digestion kinetics with plasmid purified using the ChargeSwitch®-Pro Kit. ChargeSwitch®-Pro Miniprep column-purified DNA (C) can be digested faster than DNA purified using a silica column from a competitive kit (Q), pUC19 (A) or pLadder (B) purified with the ChargeSwitch®-Pro kit were completely digested in about 30 min, whereas the silica-purified sample was only partly digested after 90 min.
Figure 5. Higher quality of supercoiled plasmid DNA. Supercoiled plasmid DNA represents a measure of both purity and how greatly a sample is treated during purification. ChargeSwitch®-Pro purified plasmid exhibits a higher degree of supercoiling versus a silica-based prep from Competitive Q, which displays both supercoiled and nicked species of plasmid DNA.