1
miniBio Column Plasmid Mini-Preps Kit
CAT. NO.: mB001
Package: 100 Preps
Kit Contents
Components
mB001，100 Preps
Spin Column & Collection Tube
100 sets
Buffer P1
28 ml
Buffer P2
28 ml
Buffer P3
40 ml
Buffer DW1
50 ml
Wash Solution
24 ml
Elution Buffer
10 ml
RNase A
4.2 mg
VisualLyse
120 μl
Protocol
1
Storage and stability
miniBio Column Plasmid Mini-Preps Kit should be stored dry, at room temperature (15-25°C) except for buffer P1, which
should be stored at 4°C after addition of RNase A.
If precipitate forms in the buffers during storage, it should be redissolved by incubating the buffers at 37°C and cool
down to room temperature before use.
Safety instructions
Buffer P2 contains sodium hydroxide thus is irritating to eyes and skin. Please avoid contact with eyes, skin, and
clothes. Wash thoroughly after handling. Keep container closed.
Buffer P3 contains acetic acid. Avoid contact with skin and eyes. Avoid ingestion and inhalation.
RNase A may cause sensitization by inhalation and skin contact.
Buffer DW1 contain isopropanol thus is flammable.
24-hour emergency information
Emergency medical information in English, French, and German can be obtained 24 hours a day from: Poison
Information Center Mainz, Germany. Tel.: +49-(0)6131-19240
Introduction
miniBio Column Plasmid Mini-Preps Kit is designed for rapid and cost-effective small-scale preparation of high quality
plasmid DNA from recombinant E. coli cultures. Pelleted bacterial cells are resuspended and subjected to SDS/alkaline lysis
to liberate the plasmid DNA. Cell debris and SDS precipitate are pelleted by centrifugation and the plasmid DNA in the
supernatant is selectively adsorbed in silica gel-based spin column under appropriate conditions. Other contaminants such
Updated:07/2015
www.minibio.de
2
as proteins, salts, nucleotides and oligos are removed by column washing. The purified plasmid DNA was then eluted with
Elution Buffer and is ready for immediate use in all molecular biology procedures such as restriction enzyme digestion, PCR,
transformation and sequencing. Each spin column can recover up to 20 μg of plasmid DNA. The kit can be successfully
used for efficient purification of plasmids and cosmids from 100 bp to 10 kb. The actual plasmid yield and optimal culture
volume depend on the plasmid copy number and medium used for cultivation.
Features
1.
Fast and effective. Entire procedure takes 15-20 minutes and 10~20 μg DNA can be harvested.
2.
Preparation of high quality plasmid DNA from bacterial cultures. Purified DNA can be used in any downstream
applications such as sequencing, PCR, transformation, restriction enzymatic digestion, and ligation.
3.
No phenol/chloroform extraction or ethanol precipitation is required.
4.
VisualLyse provide a color change during the lysis and neutralization step, it prevents inefficient cell lysis and
precipitation of SDS and proteins.
Materials Supplied by User
Microcentrifuge capable of 13,000 rpm (~17,900 x g)
Sterile 1.5 ml or 2 ml centrifuge tubes
Absolute ethanol (96%-100%)
Before Starting
It is strongly advised that you read this manual thoroughly before starting. miniBio Column Plasmid Mini-Preps Kit is
designed to be simple, fast and reliable provided that all steps are followed diligently. Prepare all components, and have the
necessary materials as outlined before starting.
RNase A is provided in lyophilized form during transportation. Before use, add 1 ml Buffer P1 to RNase A, dissolve
completely, and then add the RNase A Solution to the bottle containing Buffer P1 and mix well. After addition of RNase A,
the Buffer P1 should be stored at 4°C.
Check the Buffer P2 and Buffer P3 for salt precipitation before each use. If necessary, redissolve the precipitate by
warming the solution at 37°C, then cool back down to room temperature before use. Do not shake the Buffer P1 too
vigorously.
Add 96 ml absolute ethanol to the 24 ml Wash Solution (volume of ethanol:volume of Wash Solution=4:1).
Elution Buffer is 2.0 mM Tris-HCl, pH 8.5. Water can be used as eluate in the final step, but it is not recommended if the
pH of water is less than 7.0.
Procedures
Notes:
 All centrifugations are carried out at 13,000 rpm (~17,900 x g) with a conventional, table-top microcentrifuge.
 All steps should be carried out at room temperature (15–25°C).
1.
Inoculate 5 ml LB medium with appropriate antibiotic placed in a 10~20 ml culture tube with E. coli harboring
the desired plasmid and grow at 37°C with vigorous shaking for 12~16 h.
2.
Pellet bacterial cells from 1.5~5 ml culture by centrifugation for 30 sec - 1 min (or as wished). Drain or aspirate
the medium completely.
Note: Do not overload cells. Otherwise excessive biomass may cause inefficient lysis resulting poor yield or DNA quality.
Updated:07/2015
www.minibio.de
3
For Low copy plasmids: Generally yield 0.1~0.5 μg DNA per ml overnight culture could be achieved. For routine screening
of recombinant clones, a 5 ml culture should provide ample material for agarose gel visualization or restriction digest
analysis. However, the method can be modified to essentially double the yield if necessary. Start with 10 ml bacterial culture,
and pellet cells either successively, 1.5 ml of culture at a time, or centrifuge for 5 min at 5000 × g in a 15 ml centrifuge tube.
Proceed to Step 3 and double the volumes of Buffer P1, P2, and P3. Continue as above using only one spin column per 10
ml culture. There is no need to increase the volume of Wash Solution used.
For high copy number plasmids: Because the spin column quickly becomes saturated with above 5 ml culture, we
recommend processing of multiple samples from the same culture.
3.
Add 250 μl of Buffer P1 to the pellet, resuspend the pellet completely by pippetting or vortexing.
Note: Complete resuspension of cell pellet is vital for obtaining good yields.
Note: VisualLyse can be used in this step, add 1 μl of VisualLyse and mix thoroughly.
4.
Add 250 μl of Buffer P2 to the mixture, mix gently but sufficiently by inverting the tube 4~6 times and then
keep at room temperature for 3~5 min.
Note: To prevent contamination from genomic DNA, do NOT vortex or mix aggressively, and the incubation time should
NOT exceed 5 min. Store Buffer P2 tightly capped when not in use.
Note: If VisualLyse was used, the solution turns blue after the addition of Buffer P2.
5.
Add 350 μl of Buffer P3, mix gently but sufficiently by inverting the tube 4-6 times, and centrifuge immediately
for 10 min.
Note: If VisualLyse was used, the solution turn colorless again, any trace of blue indicates insufficient mixing.
6.
Carefully transfer all the clear supernatant to a clean spin column assembled in a 2 ml collection tube
(provided). Ensure that the pellet is not disturbed and that no cellular debris is carried over into the column.
Centrifuge for 30 sec for lysate to completely pass through column.
Note: If the volume of lysate exceeds 700 μl, load 700 μl to the column, centrifuge for 30 sec and discard liquid. Load the
remaining lysate to the same column and centrifuge as above.
7.
(Optional) Discard liquid in the collection tube, put the column back in the same collection tube, add 500 μl
Buffer DW1 to the column and centrifuge for 30 sec.
Note: This step is optional. Buffer DW1 serves as a deproteinization buffer, which is NOT necessary for endA- bacteria like
DH5α, BUT is highly recommended for endA+ bacteria such as BL21, however.
8.
Discard the flow-through, put the column back in the same collection tube, add 500 μl Wash Solution to the
column and centrifuge for 30 sec.
Note: Wash Solution must be diluted with absolute ethanol before use. See label for directions.
9.
Repeat wash procedure in step 8 once.
10. Discard the flow-through and centrifuge the empty column for 1 min.
Note: This step is important for removing residual ethanol, which may inhibit subsequent enzymatic reactions. Keep the
column at room temperature for 5-10 minutes after this step can further remove the ethanol.
Updated:07/2015
www.minibio.de
4
11. Transfer the column to a clean 1.5 ml microfuge tube. Add 50~100 μl (depending on desired concentration of
final product) Elution Buffer (2 mM Tris-HCl, pH 8.5) or water to the center of the membrane, incubate 1 min at
room temperature and centrifuge for 1 min to elute DNA. Store the purified DNA at -20°C.
Note: To gain higher plasmid DNA yield or for large plasmids, heat the Elution Buffer to 60°C before apply to the spin column
and extend the incubation time to 5 min.
12. Yield and quality of DNA: Determine the absorbance of the sample at 260 nm and then at 280 nm. Alternatively,
quantity and quality can sometimes best be determined by agrose gel/ethidium bromide electrophoresis.
Typically, the majority of the DNA eluted is in monomeric supercoil form, though other bands may also be
present.
Troubleshooting Guide
Problem
Possible Causes
Possible Solutions
Low DNA yields
Incomplete lysis of
Overload of cells. Do not use more than 5 ml culture with protocol and
bacterial cells
drain the medium completely prior to the addition of Buffer P1.
Cell pellet not properly resuspended. Make sure to vortex cell
suspension to completely disperse.
Invalidation of Buffer P2. Buffer P2 should be tightly closed
immediately after use or freshly prepared as follows: 0.2 M NaOH, 1%
SDS.
Bacterial clone is overgrown or not fresh. Do not incubate cultures for
more than 16 hr at 37°C.
Low-copy number
Increase culture volume to 10 ml and increase the volume of Buffer P1,
plasmid used
P2 and P3 accordingly. Load the lysate on the same column.
Low elution efficiency
Suboptimal elution conditions. The pH of Elution Buffer or Water must
be ≥ 8.0. A longer incubation time with Elution Buffer (up to 10 minutes)
or higher elute temperature (60°C) can improve the yield for large
plasmids.
No DNA Eluted
Improper Wash
Wash Solution concentrate not diluted with ethanol. Prepare Wash
Solution
Solution according to instructions.
Contamination of
Over treated with
Over mixing of cell lysate upon addition of Buffer P2. Do not vortex
chromosomal DNA
Buffer P2
or mix aggressively after adding Buffer P2. Also, the period of lysis
should not be longer than 5 min.
Sample contains RNA
Deficient RNase
Too many cells were harvested. We recommend using the appropriate
activity
volume according to the protocol.
RNase A not added to Buffer P1 or RNase activity is
weakened. Buffer P1 with RNase A should be stored at 4°C for less than
6 months.
Restriction enzyme
Ethanol residue
inhibition
Ethanol has not completely been removed from column following
wash steps. Centrifuge column as instructed to dry the column before
elution.
Co-elution of glass
Co-eluted glass fibers may inhibit restriction enzyme. Centrifuge for
fibers
1 min at maximum speed and transfer supernatant to a new tube.
Updated:07/2015
www.minibio.de
5
----------Detach this page to have a Short Protocol for Experienced User----------
Notes:
 All centrifugations are carried out at 13,000 rpm (~17,900 x g) with a conventional, table-top microcentrifuge.
 All steps should be carried out at room temperature (15–25°C).
miniBio Column Plasmid Mini-Preps Procedures in Short:
1.
Pellet cells from 1.5~5 ml overnight culture by centrifugation for 30 sec.
2.
Resuspend cells in 250 μl Buffer P1 with 100 μg/ml RNase A by pippetting or vortexing.
3.
Add 250 μl Buffer P2. Mix gently by inverting 4~6 times and incubate for 3~5 min to obtain clear lysate.
4.
Add 350 μl Buffer P3 and mix well by inverting gently 4~6 times to form white precipitate. Centrifuge immediately for
10 min.
5.
Transfer the clear lysate to a Spin Column placed in a 2 ml collection tube carefully. Centrifuge for 30 sec.
6.
(optional, highly recommended for endA+ bacteria such as BL21.) Discard flowthrough, put the column back in the
same collection tube, add 500 μl Buffer DW1 to the column and centrifuge for 30 sec.
7.
Discard flowthrough, put the column back in the same collection tube, add 500 μl Wash Solution to the column and
centrifuge for 30 sec.
8.
Repeat step 7.
9.
Discard flowthrough and centrifuge empty column for 1 min to remove residual ethanol.
10. Put the column onto a new 1.5 ml tube, add 50~100 μl Elution Buffer to the center of the membrane, incubate 1 min
and centrifuge for 1 min. Store the eluted DNA at -20°C.
Updated:07/2015
www.minibio.de