Apr 04, 2018
Version 3
Plasmid DNA miniprep protocol for EZ-10 Spin Column Plasmid DNA minipreps kit (BS413), 50 preps V.3
- Joshua Lucate1
- 1Bio Basic Inc.
- Bio Basic Inc.
Protocol Citation: Joshua Lucate 2018. Plasmid DNA miniprep protocol for EZ-10 Spin Column Plasmid DNA minipreps kit (BS413), 50 preps. protocols.io https://dx.doi.org/10.17504/protocols.io.n8tdhwn
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: Working
We use this protocol and it's working
Created: April 04, 2018
Last Modified: April 04, 2018
Protocol Integer ID: 11251
Abstract
This protocol is for the extraction and purification of up to 10ug of plasmid DNA. It can be used for the purification of plasmid DNA ranging from 40 bp to 40kbp.
Guidelines
Introduction
The EZ-10 Spin Column Kits provide a simple and efficient method for purification of plasmid DNA, extraction of DNA from agarose gels, and purification of DNA from enzymatic reactions such as PCR or restriction enzyme digestions.
The DNA is selectively adsorbed in silica gel-based EZ-10 column and other components are washed away. The DNA is then eluted off the column and can be used for any downstream applications.
The purification method used in these protocols does not require use of phenol, chloroform, or CsCl. The DNA is purified without an additional step of ethanol precipitation.
Limitations of Use
These kits are designed for research use only. The purified plasmid DNA should not be used for live animal transfections. It is also not to be used for human diagnostic or drug production purposes.
Features
- Simple, Fast and Efficient
- Preparation of high quality DNA which can be used in any downstream applications such as sequencing, PCR, transformation or restriction digestions
- HighYield and Reproducible
- High Capacity -Up to 10μg of DNA per column
Storage
EZ-10 Spin Column Kits should be stored dry at room temperature (15 °C-25 °C). Kits can be storedfor up to 24 months without showing any reduction in performance and quality. RNase A stock solution can be stored for 2 years at 4 °C. After addition of RNase A, Solution I is stable for 6 months at 4 °C.
Quality Control
Each lot of EZ-10 Spin Column kit is tested against predetermined specifications to ensure consistent product quality.
Principle:
This kit provides a simple and efficient method formini plasmid DNA purification. The plasmid DNA is selectively adsorbed in silica gel-based EZ-10 column and other impurities such as proteins, salts,nucleotides, oligos (<40-mer) are washed away. In order to maximize the recovery yield of plasmid DNA, a color indicator-VisualLyse is added to the buffer which prevents insufficient or over-lysis during lysis and neutralization step. The plasmid DNA is then eluted off the column and can be used for any downstream application.
Materials
MATERIALS
EZ-10 Spin Column Plasmid DNA Miniprep KitBio Basic Inc.Catalog #BS413.SIZE.50preps
1.5ml Tube, Natural, 1000/BagBio Basic Inc.Catalog #BT620-N.SIZE.1PK
Ethanol, anhydrous (85%)Bio Basic Inc.Catalog #D0193.SIZE.500ml
STEP MATERIALS
1.5ml Tube, Natural, Sterile, 1000/BagBio Basic Inc.Catalog #BT620-NS.SIZE.1PK
1.5ml Tube, Natural, Sterile, 1000/BagBio Basic Inc.Catalog #BT620-NS.SIZE.1PK
Protocol materials
1.5ml Tube, Natural, Sterile, 1000/BagBio Basic Inc.Catalog #BT620-NS.SIZE.1PK
In Materials, Materials, Step 11
EZ-10 Spin Column Plasmid DNA Miniprep KitBio Basic Inc.Catalog #BS413.SIZE.50preps
Materials
1.5ml Tube, Natural, 1000/BagBio Basic Inc.Catalog #BT620-N.SIZE.1PK
Materials
Ethanol, anhydrous (85%)Bio Basic Inc.Catalog #D0193.SIZE.500ml
Materials
Before start
1) Add RNAse A solution to the bottle containing Solution 1 and mix well. Once RNAse A solution is added to solution 1, the resulting solution is stable for 6 months at 4oC. If being used infrequenly the solution can be stored at -20 oC for longer periods as long as freeze thaw cycles are minimized.
2) If solution 2 contains a precipitate, dissolve the precipitate before use by gently warming the solution at 37 oC.
3) Before using the wash solution, add 80 mL of 96-100% Ethanol to the 20 mL wash solution. Anhydrous Ethanol from Bio Basic Inc. (D0193) is suitable for use with this protocol.
Add 1.5 mL of overnight culture to a microcentrifuge tube and centrifuge at 12,000rpm for 2 minutes. Drain the clarified supernatant completely leaving only the cell pellet.
Optional: Depending on ease of plasmid replication add a further 1.5 mL of overnight culture to the microcentrifuge tube containing the previous pellet and repeat until a maximum of 5 mL of overnight culture has been added.
00:02:00
Add 100uL of Solution 1 to the pellet, mix well, ensuring that no clumps are present and let sit for 1 min
00:01:00
Add 1μL of VisualLyse to the solution from step 2 (optional)
Add 200µl of Solution II to the mixture, and mix gently by inverting the tube 4-6 times and then keep at room temperature for 1 minute. To prevent contamination from genomic DNA, do not vortex.
00:01:00
Note
If VisualLyse has been added, the solution will turn blue after addition of Solution II. A homogenously blue suspension should then be observed. If the suspension contains uneven blue color, or white/brownish cell clumps, continue mixing carefully.
Add 350µl of Solution III, and mix gently. Incubate at room temperature for 1 minute. A fluffy white material forms and lysate should become less viscous. If VisualLyse has been added in step 3, the suspension should be mixed until all traces of blue has gone and lysate becomes colorless.
00:01:00
Centrifuge at 12,000rpm for 5 minutes
00:05:00
Transfer the above supernatant (step 6) to the EZ-10 column. Centrifuge at 10,000rpm for 2 minutes
00:02:00
Discard the flow-through in the tube. Add 750µl of Wash Solution to the column, and centrifuge at 10,000rpm for 2 minutes
00:02:00
Repeat wash procedure in step 8
Discard the flow-through in the collection tube. Centrifuge at 10,000rpm for an additional minute to remove any residual Wash Solution
00:01:00
Transfer the column to a clean 1.5ml microfuge tube (BT620-NS). Add 50µl of Elution Buffer into the center part of the column and incubate at room temperature for 2 minutes.
00:02:00
Note
Note: It is extremely important to add the Elution Buffer into the center part of the column.
Incubating the column with the Elution Buffer at higher
temperature (37ºC to 50ºC) may slightly increase the yield especially for large (>10,000bp) DNA Plasmids. Prewarming the Elution Buffer at 55ºC to 80ºC may also slightly increase elution efficiency.
1.5ml Tube, Natural, Sterile, 1000/BagBio Basic Inc.Catalog #BT620-NS.SIZE.1PK
Centrifuge at 10,000 rpm for 2 minutes
00:02:00
Store purified DNA at -20ºC