About ZR Plasmid Miniprep™ - Classic
The ZR Plasmid Miniprep™-Classic is designed for efficient isolation of plasmid DNA from E. Coli using a traditional 3-buffer (P1, P2, P3) procedure that is simple, rapid, user-friendly, and reliable. It features a modified alkaline lysis protocol together with a unique Fast-Spin column to yield high quality plasmid DNA in minutes. The buffers are color-coded (red, green, yellow) for easy determination of complete cell lysis and neutralization. The innovative Zymo-Spin™ llN columns yield endotoxin-free plasmid DNA. Plasmid DNA purified using the ZR Plasmid Miniprep™-Classic is well suited for use in restriction endonuclease digestion, sequencing, DNA ligation, cloning, PCR, bacterial transformation, transfection, etc.
|DNA Recovery||≥ 30 µl|
|Processing Time||15 min|
|DNA Purity||Plasmid DNA which is well suited for ligation, sequencing, restriction endonuclease digestion, in vitro transcription, and other sensitive applications requiring pure DNA. Typical A(260/280) ≥ 1.8.|
|Plasmid DNA Size||Up to 25 kb.|
|Plasmid DNA Yield||Up to 25 µg per preparation, depending on the plasmid copy number, culture growth conditions, and strain of E. Coli utilized.|
|Procedure||Performed at room temperature (15-30°C) using a centrifuge and/or a vacuum manifold.|
Plasmid products. Restriction Endonuclease digestion of three different plasmids prepared using the ZR Plasmid Miniprep™-Classic, performed in triplicate. M: ZR 1 kb DNA marker (Zymo Research)
Sequence-quality DNA preparations. DNA sequencing chromatogram of plasmid DNA prepared using the ZR Plasmid Miniprep™-Classic.
Centrifuge 0.5 - 5 ml1,2 of bacterial culture in a clear 1.5 ml tube at full speed for 15 - 20 seconds in a microcentrifuge. Discard supernatant.
Add 200 µl of P1 Buffer (Red) to the tube and resuspend pellet completely (i.e., by vortexing or pipeting).
Add 200 µl of P2 Buffer (Green)3 and mix by inverting the tube 2 - 4 times. Cells are completely lysed when the solution appears clear, purple, and viscous. Proceed to the next step within 1-2 minutes.
Add 400 µl of P3 Buffer (Yellow) and mix gently but thoroughly. Do not vortex. The sample will turn yellow when the neutralization is complete4. Allow the lysate to incubate at room temperature for 1-2 minutes.
Centrifuge sample(s) for 2 minutes.
Place a Zymo-Spin™ IIN column in a Collection Tube and transfer the supernatant from Step 5 into the Zymo-Spin™ IIN column. When pipeting the supernatant be careful not to disturb the green pellet to avoid transferring any cellular debris to the column.
Centrifuge the Zymo-Spin™ IIN/Collection Tube assembly for 30 seconds.
Discard the flow-through in the Collection Tube, making sure the flow-through does not touch the bottom of the column. Return the Zymo-Spin™ IIN column to the Collection Tube5.
Add 200 µl of Endo-Wash Buffer to the column and centrifuge for 30 seconds.
Add 400 µl of Plasmid Wash Buffer6 to the column. Centrifuge for 1 minute.
Transfer the column into a clean 1.5 ml microcentrifuge tube and then add 30 µl (of DNA Elution Buffer7 to the column. Centrifuge for 30 seconds to elute the plasmid DNA.