TITLE: E.Z.N.A.® Plasmid DNA Maxi Kit Centrifugation Protocol (modified)
AUTHORS: Bettina Ergün

[DESCRIPTION]
Describes a modified protocol to use the E.Z.N.A.® Plasmid DNA Maxi Kit # D6922 from Omega Bio-Tek without  Ultracentrifuge.
For additionally information read the manufacturer's manual (attached)

[STEPS]
SECTION: Extraction and purification
4. Transfer 50-200 mL overnight culture to an appropriate centrifuge tube

Note: The optimal volume to use depends on the culture density and plasmid copy number. The optimal cell mass (OD600 x mL culture) for the HiBind‱ DNA Maxi Column is 300-400. For example, if the OD600 of a culture is 4.0, the optimal culture volume should be 75-100 mL. If excess culture cell mass is used, alkaline lysis will be inefficient, the HiBind‱ membrane will be overloaded, and the performance of the system will be decreased. The increase in lysate viscosity will require vigorous mixing which may result in shearing of genomic DNA and contamination the plasmid DNA.
SECTION: Extraction and purification
5. Centrifuge at 4,000g for 10 minutes.
SECTION: Extraction and purification
6. Decant or aspirate and discard the culture media.

Note: To ensure that all traces of the medium are removed, use a clean paper towel to blot excess liquid from the wall of the tube.
SECTION: Extraction and purification
7. Add 12 mL Solution I/RNase A. Vortex or pipet up and down to completely resuspend the cells.

Note: RNase A must be added to Solution I before use.
SECTION: Extraction and purification
8. Transfer the cell suspension to a 50 mL centrifuge tubes.
SECTION: Extraction and purification
9. Add 12 mL Solution II. Invert and rotate the tube 2-3 minutes on a IKA rocker 3D at 25 rpm to obtain a cleared lysate.

Note: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 minutes. Store Solution II tightly capped when not in use to avoid acidification from CO2 in the air.
SECTION: Extraction and purification
10. Add 16 mL Solution III. Invert and rotate the tube 2-3 minutes on a IKA rocker 3D at 25 rpm until flocculent white precipitates form.

Note: It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation.
SECTION: Extraction and purification
11. Centrifuge at 4258g (max. g!) for 30 minutes at 4°C. Otherwise use a Ultracentrifuge at 15000g for 10 min (Original protocol) A compact white pellet will form. Promptly proceed to the next step.
SECTION: Extraction and purification
12. Insert a HiBind‱ DNA Maxi Column into a 50 mL Collection Tube (provided).
SECTION: Extraction and purification
14. Centrifuge at 4,000g for 5 minutes.
SECTION: Extraction and purification
12.1. Optional Protocol for Column Equilibration:
Add 3 mL 3M NaOH to the HiBind® DNA Maxi Column.
Let sit at room temperature for 4 minutes.
Centrifuge at 4,000g for 3 minutes.
Discard the filtrate and reuse the collection tube.
SECTION: Extraction and purification
13. Transfer 20 mL cleared supernatant from Step 8 by CAREFULLY pipetting it into the HiBind‱ DNA Maxi Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind‱ DNA Maxi Column.
SECTION: Extraction and purification
15. Discard the filtrate and reuse the collection tube.
SECTION: Extraction and purification
16. Repeat Steps 10-12 until all of the cleared supernatant has been transferred to the HiBind‱ DNA Maxi Column.
SECTION: Extraction and purification
17. Add 10 mL HBC Buffer.

Note: HBC Buffer must be diluted with 100% isopropanol prior to use. Please see the instructions in the “Preparing Reagents” section on Page 6.
SECTION: Extraction and purification
18. Centrifuge at 4,000g for 5 minutes.
SECTION: Extraction and purification
19. Discard the filtrate and reuse the collection tube.
SECTION: Extraction and purification
20. Add 15 mL DNA Wash Buffer.
Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use.
SECTION: Extraction and purification
21. Centrifuge at 4,000g for 5 minutes.
SECTION: Extraction and purification
22. Discard the filtrate and reuse the collection tube.
SECTION: Extraction and purification
23. Repeat Steps 20-22 for a second DNA Wash Buffer wash step.
SECTION: Extraction and purification
24. Centrifuge the empty HiBind‱ DNA Maxi Column at 4,000g for 10 minutes to dry the column matrix.

Note: It is important to dry the HiBind‱ DNA Maxi Column matrix before elution. Residual ethanol may interfere with downstream applications.
SECTION: Extraction and purification
25. Transfer the HiBind‱ DNA Maxi Column to a nuclease-free 50 mL centrifuge tube (not provided).
SECTION: Extraction and purification
26. Add 1.5-3 mL Elution Buffer or sterile deionized water directly to the center of the column membrane.
SECTION: Extraction and purification
27. Let it sit at room temperature for 5 minutes.

Note: This represents approximately 65-80% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration. Alternatively, a second elution may be performed using the first eluate to maintain a high DNA concentration.
SECTION: Extraction and purification
28. Use the first eluat for a second elution step.
SECTION: QC and storage
29. Qubit Concentration determination as described in separate protocol.
SECTION: QC and storage
30. Aliquot and store DNA at -20°C.
SECTION: Before Start
2. Check Solution II and Solution III for precipitation before use. Redissolve any precipitates by warming to 37°C
Optional: Heat Elution Buffer to 65°C if plasmid DNA is >10 kb
All centrifugation steps should be carried out at room temperature unless otherwise noted.
SECTION: QC and storage
29.1. place here the concentrations and the aliquotation of the samples
SECTION: Samples
3. place here the informations on the samples


SECTION: Before Start
1. Prepare DNA Wash Buffer, HBC Buffer, and Solution I:
Add vial of RNase A to the bottle of Solution I provided and store at 2-8˚C.
Dilute DNA Wash Buffer with 100% ethanol as writen on the bottle and store at room temperature.
Dilute HBC Buffer with 100% isopropanol  as writen on the bottle and store at room temperature.