Part of the D420 ZymoPURE Plasmid Kits collection, the D420 ZymoPURE II Plasmid Midiprep kit provides the fastest and simplest method available to efficiently isolate up to 1.2 mg of transfection grade plasmid DNA from E. coli. Utilizing modified alkaline lysis in conjunction with a patented binding system, the ZymoPURE II Midiprep kit can process up to 50 ml of culture in less than 18 minutes. This remarkable kit results in significantly more plasmid DNA while providing drastically reduced processing times. The plasmid DNA is rapidly bound onto a column with either a vacuum or a centrifuge instead of a slow gravity flow column. Additionally, there is no alcohol precipitation step required and the elution is performed using a microcentrifuge. The recovered plasmid DNA is highly concentrated (up to 6 µg/µl), endotoxin-free and transfection-ready. As an added convenience, the D420 ZymoPURE II Plasmid Midiprep kit contains colored buffers that permit error-free visualization and identification of complete bacterial cell lysis and neutralization.
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| Applicable For | Transfection, transformation, lentivirus production, adenovirus production, AAV production, CRISPR, genome editing, in vivo studies, sequencing, restriction endonuclease digestion, in vitro transcription/translation, PCR, and other sensitive applications. |
|---|---|
| Binding Capacity | 1.2 mg |
| Culture Input | ≤ 50 ml |
| Elution Volume | ≥ 150 µl |
| Endotoxin Levels | ≤ 0.025 EU/µg of Plasmid DNA |
| Equipment | Microcentrifuge and vacuum/vacuum manifold (recommended) or swinging bucket centrifuge |
| Format | Spin-Column |
| Processing Time | ≤ 18 min |
| Purity | Typical Abs 260/280 ≥1.8 and Abs 260/230≥ 2.0 |
| Size Range | Up to 200 kb |
| Yield | Up to 1.2 mg per preparation. Actual yield is dependent on the plasmid copy number, culture growth conditions, and strain of E. coli utilized. Typical yields from 50 ml of overnight culture grown in LB for high copy number plasmids are 200 – 400 µg. |
Q1: What is the composition of the ZymoPURE Elution buffer?
10 mM Tris-HCl, 0.1 mM EDTA, pH 8.5.
Q2: Can the ZymoPURE Kits be used with other bacteria?
Yes, the standard protocol should work with other gram-negative bacteria. However, the user will need to validate the protocol for their particular species. For gram-positive bacteria, please refer to the Gram-Positive Bacteria Protocol in the Appendix of the kit protocol.
Q3: What type of vacuum pump do you recommend?
The vacuum pump should be a single or double-staged unit capable of producing at least 400 mm Hg pressure at the vacuum manifold. If less pressure is applied, centrifuge the column prior to washing to remove any residual lysate/buffer remaining in the matrix.
Q4: Are the ZymoPURE kits compatible with any commercially available vacuum manifold?
Yes, any vacuum that uses standard luer-lock connectors is compatible.
Q5: Can an in-house vacuum line be used with the EZ-Vac Vacuum Manifold?
Yes, however, the pressure needs to be around 400 mm Hg for the liquid to pass through the columns quickly. Users should take caution, as the pressure of an in-house vacuum line can fluctuate drastically or be significantly reduced, depending on the demand in the building.
Q6: I ran out of ZymoPURE Wash 2. Can I substitute it with a homemade solution or Wash Buffer from another kit?
No, the ZymoPURE kits are only compatible with ZymoPURE Wash 2 and we cannot disclose a substitution due to the sensitive nature of the recipe. Additional ZymoPURE Wash 2 can be purchased separately.
Q7: Is there a protocol for low-copy number plasmid DNA?
Yes, the low-copy protocol can be found in the Appendix of the kit protocol.
Q8: Can I exceed the recommended volume of bacterial culture or use enriched growth media?
Yes, however, special care should be taken throughout the entire protocol since there is more biomass. We recommend increasing the incubation time during lysis and centrifuging the neutralized lysate before loading onto the supplied syringe filter. Exceeding the recommended culture volume or using enriched media for high copy plasmids may overload and subsequently clog the columns, which can reduce DNA yield/quality or result in failed preps.
Q9: Can the ZymoPURE kits be used to isolate large plasmid constructs (BAC/PAC)?
Yes, the standard ZymoPURE protocol has been successfully tested with constructs up to 200 kb. To increase the elution efficiency of large plasmid DNA, we recommend pre-warming the ZymoPURE Elution Buffer (50 ºC) and increasing the incubation time on the column up to 10 minutes prior to centrifugation.
Q10: I accidently left my ZymoPURE P1 at room temperature, is it still okay to use?
Yes, the RNase A is fairly stable at room temperature, but we recommend placing it in 4 °C as soon as possible to ensure optimal performance throughout the life span of the product.
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