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Super-pure 96 Plasmid DNA Purification Kit |
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Kit contents, storage and stability |
| Cat.
No |
10-210163-04 |
10-210163-18 |
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Preparation
96-well
Block (square, 2.2 ml)
96-well Filter Plate
96-well DNA-prep Plate
96-well V-shaped Bottom Plate
500-ml Bottle
Adhesive Tape Sheet
R Nase A1
Buffer S1
Buffer S2
Buffer S3
Buffer W1
Buffer
W2
10×Buffer W2
Eluent
Manual
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4×96
4
4
4
4
---
32
270 μl
135 ml
135 ml
200 ml
185 ml
100 ml×3
---
60 ml
1
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18×96
18
18
18
18
2
144
620 μl×2
310 ml×2
310 ml×2
460 ml×2
410 ml×2
---
330 ml
250 ml
1
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The
Kit can be stored for at least one year without showing reduction
in the efficiency of isolation.
RNase A1: 50 mg/ml, store at -20℃.
Buffer S1: Bacterial suspension buffer. Store at 4℃ after addition
of RNase A1.
Buffer S2: Cell lysis buffer. Store at room temperature.
Buffer S3: Neutralization buffer. Store at room temperature.
Buffer W1: Wash buffer. Store at room temperature.
Buffer W2: Desalting buffer. Before use, add
ethanol as much as indicated on the bottle, and mix well. Store
at room temperature.
10×Buffer W2: Desalting buffer. Store at room
temperature. Before use, place 1-volume 10×Buffer W2 to a 500-ml
Bottle, add 3.5-volume dH2O and 5.5-volume absolute ethanol, and
mix well. For example, 50 ml of 10×Buffer W2 is placed to 500-ml
Bottle, then add 175 ml of dH2O and 275 ml of absolute ethanol.
Eluent: 2.5mM Tris-HCl, pH8.5. Store at room temperature.
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Introduction |
Super-pure 96 Plasmid DNA Purification Kit is suitable for isolation
of plasmid DNA up to 6 μg from 1.3-ml high-copy-plasmid-containing
bacterial culture. For isolation of low-copy plasmid containing pMB1
or ColE1, the culture should be added with chloramphenicol at final
concentration of 170 mg/ml and incubated for another 20～24 hours to
increase the copies of plasmid.
The purification of plasmid with this Kit is based on modified alkaline
lysis and selectively binding of DNA to silica membrane. The purified
plasmid DNA is free from contamination with protein, genomic DNA and
RNA, and is suitable for large-scale sequencing, in vitro transcription
and translation, screening based on restriction enzyme digestion,
PCR amplification, and eukaryotic transfection, etc.
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Principle
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1.
Cells lysis and neutralization
Bacterial cells are lysed in NaOH/SDS (Buffer S2). After addition
of Buffer S3, protein is denatured and co-precipitated with genomic
DNA. While, plasmid DNA is re-natured, forms compact super-coiled
configuration and remains in the supernatant. Meanwhile, Buffer
S3 contains high concentration DNA-binding salt, which facilitates
DNA to bind efficiently to silica membrane.
2. Removal of impurities by filtration and
selectively binding of DNA
96-well Filter Plate can be assembled with 96-well DNA-prep Plate
in order to simplify purification procedure and save laboratory
labour. Applying the assembled set on Vacuum Manifold (see Figure
1 on page 4), removal of impurities and binding of DNA can be completed
simultaneously.
(1) The 96-well Filter Plate contains two layers of filtration membranes.
The rough-filtration membrane entraps large particles to increase
the filtration efficiency of underlined fine-filtration membrane
with pore size of 1 μm, which ensures to remove tiny particles to
prevent capillary from being blocked.
(2) The 96-well DNA-prep Plate contains a silica membrane characterized
by selective adsorption of DNA in a buffer containing certain salt
and elution in water or low-salt buffer. The optimized buffers used
in this Kit ensure that only DNA will be adsorbed onto silica membrane,
while RNA, cellular proteins and metabolites are not retained on
the membrane.
3. Washing to remove impurities
The impurities on the membrane are removed by washing with Buffer
W1, followed with Buffer W2 to remove salt. To accelerate evaporation
speed of ethanol contained in Buffer W2, the DNA-prep Plate is washed
once with absolute ethanol.
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Description
of protocol |
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1.
Bacterial Culture
This Kit is designed for isolation of high-copy plasmid from 96-well
bacterial culture. For isolation of low-copy plasmid containing
pMB1 or ColE1, the culture should be added with chloramphenicol
to increase the copies of plasmid. Please refer to the following
table for details.
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DNA
construct
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Origin
of replication
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Copy
number
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Classification
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| Plasmids
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| pUC |
ColE1 |
500～700
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high copy |
| pBluescript
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ColE1 |
300～500 |
high copy |
| pTZ |
pMB1 |
>1000
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high copy |
| pBR322 |
pMB1 |
15～20 |
low copy |
| Cosmids
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| uperCos |
ColE1 |
10～20 |
low copy |
| pWE15 |
ColE1 |
10～20 |
low copy |
Bacterial cultures
for plasmid isolation should always be grown from a single colony
picked from a freshly streaked selective plate. A single colony
should be inoculated into rich media (such as TYGPN, TB, 2×TY) containing
the appropriate selective agent. For bacterial culture, the Kit
offers 96-well Block (square, 2.2 ml) with maximal culture volume
of 1.3 ml. The 96-well Block (square,
2.2 ml) should be covered with plastic lid provided with the Kit
for good aeration. Most bacteria can reach to late log phase when
grown at 37℃ with shaking at 260~300 rpm for 20～24 hours. For isolation
of low-copy plasmid containing pMB1 or ColE1, the culture should
be incubated for another 20～24 hours to increase the copies of plasmid.
2. Collection of bacterial cells
It is recommended to centrifuge at 1500±200×g to pellet bacterial
cells. At this speed bacterial cells can not only be efficiently
precipitated but also be easily re-suspended in Buffer S1. Precipitation
at high-speed will result in hard re-suspending.
3. Cells lysis and neutralization
After harvested and resuspended, the bacterial cells are lysed in
NaOH/SDS (Buffer S2). Under alkaline condition SDS can efficiently
solubilize the phospholipid and protein components of the cell membrane,
leading to lysis of bacteria, release of the cell contents and denaturation
of the chromosomal and plasmid DNA, as well as proteins. The lysis
procedure should not last over 5 min. Long term of exposure to alkaline
conditions may cause plasmid to become irreversibly denatured.
The lysate is re-neutralized and adjusted to high-salt DNA binding
conditions after addition of Buffer S3. In the neutralized solution,
chromosomal DNA, cellular debris and SDS form co-precipitate, while
the plasmid DNA is re-natured and remains in solution.
To prevent contamination of plasmid DNA with chromosomal DNA, vigorously
stirring and vortexing must be avoided during lysis. Vigorous treatment
during the lysis and neutralization procedure will shear the bacterial
chromosome, leaving free chromosomal DNA fragments in the supernatant.
Such released chromosomal fragments are chemically indistinguishable
from plasmid DNA, and will be co-purified with plasmid DNA.
4. Removal of impurities by filtration and
selective binding of DNA
The Vacuum Manifold can be set in advance (see Figure 1 on page
4). Place a 96-well Filter Plate on a 96-well DNA-prep Plate in
the same orientation, and seal the gap between the two plates. Place
the assembled set on Vacuum Manifold, transfer the neutralized solution
to 96-well Filter Plate. Applying vacuum, particle impurities is
retained in 96-well Filter Plate, and DNA in the flow-through is
binding on silica membrane in 96-well DNA-prep Plate directly.
5. Wash to remove impurities
The 96-well DNA-prep Plate is washed in turn with Buffer W1, Buffer
W2, and absolute ethanol to remove cell components, salt and water.
After washing, make sure ethanol is evaporated completely, otherwise,
residual ethanol may inhibit subsequent enzymatic reactions.
6. Elution of plasmid DNA
Plasmid binding on silica membrane can be eluted in water or Elution.
The elution volume can be determined by required concentration of
plasmid DNA. But the elution volume should not be less than 70 μl.
Apparatus needed
1. 8-channel pipette or 12-channel pipette.
2. Vessel used to contain buffers.
3. Vortex.
4. Vacuum Manifold and Vacuum Pump.
5. Centrifuge with a rotor for 96 well plates (e.g., Sigma ? 6K10,
or Heraeus Minifuge ? GL) used to collect bacterial cells and elute
plasmid DNA.
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Preparation
before experiment |
1. Prepare rich media and selective agents.
2. Add one tube of RNase A1 into one bottle of Buffer S1 and mix well.
3. Before the use of the kit, add ethanol into Buffer W2 as much as
indicated on the bottle, or dilute 10×Buffer W2 as indicated on the
bottle, and mix well.
4. Before use, check Buffer S2 for precipitation. If precipitation
occurs, incubate in water bath at 37℃ until precipitate
is dissolved, and chill to room temperature.
5. Prepare paper towel with long fiber and parafilm.
6. Prepare absolute ethanol.
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Notes |
Buffer S2, Buffer S3 and Buffer W1 contain irritant compound. When
working with the buffers, always wear suitable protective clothing
such as laboratory overalls, safety glasses and gloves. Take care
to avoid contact with skin or eyes. In the case of contact with skin
or eyes, wash immediately with water. If necessary, ask for medical
assistance.
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Protocol |
[Bacterial culture and collection]
1. Fill each well of 96-well Block (square, 2.2 ml) with 1.3 ml of
rich medium containing the appropriate selective reagent. Inoculate
each well from a single bacterial colony and cover the block with
plastic lid provided with the kit. Incubate the cultures for 20～24
hours at 37℃ with shaking at 260~300 rpm.
2. Centrifuge at 1500×g for 5 min to pellet bacterial cells. Discard
the supernatant by inverting 96-well Block (square, 2.2 ml) quickly.
Place 96-well Block (square, 2.2 ml) upside down on a paper towel
for minutes to drain off the residual medium.
[Cells lysis and neutralization]
* Before the use of the kit, add one tube of
RNase A1 into one bottle of Buffer S1, mix well and store at 4℃.
3. Add 0.3 ml
of Buffer S1 containing RNase A1 to each well, and seal the Block
with Adhesive Tape Sheet. Suspension should be complete, leaving
no bacterial clumps.
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Ensure that RNase A1 has been added to Buffer S1.
4. Discard Adhesive
Tape Sheet. Add 0.3 ml of Buffer S2 to each well, seal the block
with Adhesive Tape Sheet. Mix gently but thoroughly by inverting
the Block for about 4~6 times. This step should be completed in
5 min.
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It is important to mix gently by inverting the 96-well Block (square,
2.2 ml). Do not shake vigorously, as this will result in shearing
of genomic DNA. If necessary, continue inverting the block until
the solution becomes viscous and clear.
* After Buffer S2 is used, close the bottle immediately to avoid
neutralization of NaOH in Buffer S2 by CO2 in the air, which will
reduce efficiency in bacterial lysis.
5. Discard the
Adhesive Tape Sheet. Add 0.45 ml of Buffer S3 to each well and seal
the block with Adhesive Tape Sheet. Mix gently but thoroughly by inverting
the block for about 10 times or until uniform precipitation is formed.
Incubate at room temperature for 5 min. Discard the Adhesive Tape
Sheet.
*
It is important to mix gently by inverting the 96-well Block (square,
2.2 ml). Do not shake vigorously, as this will result in shearing
of genomic DNA.
* Before filtration in step7, it is recommended to centrifuge at
3000×g for 10 min at 4℃.
Precipitation by centrifugation will lead to quick and uniform filtration
of the supernatant and give rise to uniform yield.
[Filtration,
binding & washing]
6. Place a 96-well Filter Plate on a 96-well DNA-prep Plate in the
same orientation, and seal the gap well between two Plates with
parafilm. Refer to the following figure, place the assembled set
on the Vacuum Manifold.
Figure1: Lysate filtration & DNA binding
7. Transfer
0.9 ml of neutralized solution in step 5 to corresponding well of
96-well Filter Plate. Switch on vacuum and turn it to maximum, draw
all solution through two plates. Keep vacuum, remove the parafilm
used to seal the two plates. Discard the 96-well Filter Plate and
keep the 96-well DNA-prep Plate on the Vacuum Manifold.
8. Add 0.4 ml of Buffer W1 to each well, and draw Buffer W1 through
the Plate.
9. Add 0.8 ml of Buffer W2 containing ethanol to each well, and
draw Buffer W2 through the Plate.
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Ensure that ethanol has been added to Buffer W2, or 10×Buffer W2
has been diluted with water and ethanol.
10. Switch off
vacuum. Take off 96-well DNA-prep Plate, and vigorously tap the
plate on long-fiber paper towel for 10 times.
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Use long-fiber paper towel, or it will result in contamination of
plasmid with tiny particles.
11. Replace
the 96-well DNA-prep Plate on Vacuum Manifold, and switch on vacuum.
Repeat Step 9.
12. Add 0.8 ml of absolute ethanol to each well, and draw it through
the Plate.
13. Repeat Step 10.
14. Replace the 96-well DNA-prep Plate on Vacuum Manifold, and apply
vacuum to maximum for 15 min at room temperature, or for 5 min under
heated air flow.
*
Residual ethanol may inhibit subsequent enzymatic reactions.
[Elution]
15. Set the Washing Set as indicated in Figure 2.
Figure2: Elution
of DNA
16. Add 70~120
μl of Eluent or water to the centre of each well of 96-well DNA-prep
Plate, and let it stand for 1 min at room temperature. Apply vacuum
at maximum for 5 min, or until there are no drops pass through.
Switch off vacuum and ventilate Vacuum Manifold slowly.
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