PowerPrep® HP Plasmid Purification Kits are designed for preparing ultrapure plasmid DNA equivalent to the gold standard of two cycles of CsCl gradient purification that is suitable for all applications. The protocol is based on a proprietary ion exchange resin that produces DNA of exceptional purity (Figure 1A).
Midi and Maxipreps employ gravity flow columns. OriGene now offers Midi and Maxiprep kits with a convenient integrated pre-filter in the columns for even greater convenience and speed (Figure 1B). Neutralized lysates can be added directly to columns containing the pre-filters, thereby combining lysate clarification and column loading into one simple step.
The procedure employs modified alkaline lysis and neutralization followed by ion exchange chromatography using a patented resin designed to provide excellent capacity, resolution and endotoxin removal. The resin has uniform particles with small pores that provide a large surface area to ensure reproducible performance, fast flow rates and high yield. Long spacer arms and high charge density ensure excellent separation from other cellular contaminants, including RNA and endotoxin. The resulting DNA is ultrapure and optimal for all applications, including those requiring the highest purity such as transfection into sensitive cell lines, automated sequencing and in vitro transcription.
DNA purified with PowerPrep® HP Kits is suitable for all applications, including even the sensitive transfections. Figure 2 demonstrates that plasmid DNA purified with OriGene’s kits can be cut with EcoRI and completely religated. The design of these products results in DNA with exceptionally high yields and low levels of endotoxin, without additional steps or expense (Table 1.) Figure 3 demonstrates that high quality DNA prepared with these kits performs exceptionally well in transfection experiments, even with difficult cell lines such as Huh1. Each lot of product is routinely tested to verify purity, integrity, and column flow rate and binding capacity.
Fig. 2 Plasmid DNA (pBluescript IIKS) was prepared using the PowerPrep® HP Plasmid Purification Kits. The DNA was digested with EcoR1 and ligated with a 1.65 kb DNA fragment. Lane 1: 1 kb Ladder Lane 2: DNA before ligation Lane 3-5: Ligation with 0.02, 0.05 and 0.1 units of T4 DNA ligase.
|Table 1||Experiment 1||Experiment 2||Experiment 3|
|PowerPrep® HP Maxiprep Kit||1055||0.1||1200||4.1||563||1.0|
|Competitor Q Maxiprep Kit||540||0.4||670||7.4||369||0.8|
Table 1 Three independent laboratories performed studies comparing the yields and endotoxin levels obtained from OriGene’s PowerPrep® HP MaxiPrep Kit and the Maxiprep kit from Competitor Q. All three studies determined that there were significantly higher yields of purified DNA using OriGene’s kits compared to Competitor Q. In addition, these researchers showed that endotoxin levels were lower in DNA purified using OriGene’s kits.
Fig. 3 This comparison of transfection efficiency with several cell lines using DNA prepared by various methods demonstrates that Marligen’s Prep (now OriGene) yields DNA that offers superior performance in transfection.
|PowerPrep® HP Midiprep Kits||PowerPrep® HP Maxiprep Kits|
|OriGene||Competitor Q||OriGene||Competitor Q|
|Total Protocol Time||2 hrs 30 mins||3 hrs 45 mins||2 hrs 18 mins||3 hrs 10 mins|
|Endotoxin Levels (EU/μg)||0.01||0.02||1.0||1.0|