Aug 21, 2020
Zymo Plasmid Miniprep - Classic - CHEM 584
- 1Brigham Young University
Protocol Citation: Ken Christensen 2020. Zymo Plasmid Miniprep - Classic - CHEM 584. protocols.io https://dx.doi.org/10.17504/protocols.io.bj5bkq2n
Manuscript citation:
https://files.zymoresearch.com/protocols/_d4015_d4016_d4054_zr_plasmid_miniprep.pdf
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: August 21, 2020
Last Modified: August 21, 2020
Protocol Integer ID: 40835
Abstract
The ZR Plasmid Miniprep-Classic kit is designed for efficient isolation of plasmid DNA from E. coli cell lysates using a 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 ZR Plasmid Miniprep-Classic features color-coded (red, green, yellow) reagents for easy determination of complete cell lysis. The Zymo-Spin llN columns facilitate high yield plasmid DNA that is endotoxin-free. Plasmid DNA purified using the ZR Plasmid Miniprep-Classic kit is well suited for use in restriction endonuclease digestion, sequencing, DNA ligation, cloning, PCR, bacterial transformation, transfection, etc.
Guidelines
1. The following procedures are carried out at room temperature. All centrifugation steps should be performed between 11,000 - 16,000 x g.
Materials
MINIPREP
MINIPREP
Centrifuge 500 µL - 5 mL of bacterial culture in a clear 1.5 ml tube at full speed for 15 - 20 seconds in a microcentrifuge. Discard supernatant.
Note
Depending on the volume of bacterial culture it may be necessary to repeat Step 1 several times. You can also centrifuge larger culture volumes (e.g., 5 mL) in a 15 mL centrifuge tube, then transfer to a microcentrifuge tube following step 2.
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) 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.
Note
Excessive lysis can result in denatured plasmid DNA formation. When processing a large number of samples, work with groups of ≤ 10 at a time.
Add 400 µL of P3 Buffer (Yellow) and mix gently but thoroughly. Do not vortex. The sample will turn yellow when the neutralization is complete. Allow the lysate to incubate at room temperature for 1-2 minutes 00:02:00 .
Note
A green precipitate consisting of K·SDS and cell debris will form. A good way to mix is to shake the tube gently several times while it is inverted.
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 pipetting 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 Tube.
Note
The capacity of the collection tube with the column inserted is 800 μl. Empty the collection tube whenever necessary to prevent contamination of the spin column with the flow-through.
Add 200 µL of Endo-Wash Buffer to the column and centrifuge for 30 seconds.
Add 400 µL of Plasmid Wash Buffer 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 Buffer to the column. Centrifuge for 30 seconds to elute the plasmid DNA.
Note
The DNA Elution Buffer contains 10 mM Tris·HCl, pH 8.5, 0.1 mM EDTA. If required, pure water can be used to elute the DNA. Add the DNA Elution Buffer directly to the center of the Zymo-Spin IIN column matrix to ensure optimal DNA elution.
TROUBLESHOOTING
TROUBLESHOOTING