核心提示：IntroductionThis cheap and simplified protocol, based on
Hansen et al., 1995, gives high yields of plasmid DNA as w
This cheap and simplified protocol, based on Hansen et al., 1995,
gives high yields of plasmid DNA as well as high purity and is
suitable for cloning, PCR, sequencing, site-directed mutagenesis and
in vitro transcription, etc. It is an extremely good method for
routine application and provides a good alternative when yields tend
to be low due to low plasmid copy number for any number of reasons .
Yields are at least 3 – 5 times higher than those obtained with
commercial plasmid purification kits and often even ten-fold higher.
Plasmid DNA can be recovered from drained liquids usually discarded
when employing commercial kits. 30 – 50% more plasmid can be recovered
if these are passed through a diatomaceous earth system.
The yield of plasmid using the protocol described here is higher than
that obtained with commercial kits even if only using low or normal
quality diatomaceous earth. From a 3 ml of E. coli culture overnight,
30-60 ‘micro’g of plasmid can be obtained at a purity of 1.8 to 2.0 .
For a 50 ml overnight culture, 500 – 800 ‘micro’g of plasmid can be
The method has been successfully employed using plasmids ranging from
about 3.0 to > 100 kb. The method basically employs two steps –
alkaline Iysis of cells and elution of DNA from a home-made
diatomaceous earth binding matrix . We used a Promega WizardTM
minicolumn , but other brands/types of columns can also be used.
Centrifugation in the protocol is carried out at 13,000 rpm on a
minifuge unless otherwise specified.
Grow a 3 ml culture of E. coli overnight containing an appropriate
Harvest the cells by centrifugation for 2 min and suspend in 300 –
500 ‘micro’l of Suspension Solution at room temperature.
Add 300 – 500 ‘micro’l of Lysis Solution, mix very gently and keep
at room temperature for about 5 min
Then add 300 – 500 ‘micro’l of Neutralising Solution. Invert gently
several times and centrifuge for at least 7 min. Fresh diatomaceous
columns should be prepared during this time . Although diatomaceous
solutions store well, the columns don’t.
Carefully transfer the supernatant and mix with approximately the
same volume of Binding Bufferin a syringe and apply the mixture to the
top of a freshly made diatomaceous earth column . Once all the
solution has been transferred, and no sooner, apply gentle suction in
the same manner as described for preparation of the column.
Add 1 ml of Washing Solution and gently drain in the same way.
Then, place the column into an eppendorf tube a) and centrifuge for at
least 3 min to make sure that all the Washing Solution is completely
removed from the column. It is necessary to repeat step 6 twice .
Place the washed and drained column into a new eppendorf tube and
add 50 ‘micro’l of preheated MQ water or TE buffer to elute the DNA
and place at room temperature for 10 min .
Centrifuge the column for 1 – 2 min. Repeating step 7 – 8 two or
three times elutes virtually all of the DNA.
a) For economic purposes, save the emptied eppendorf tube from step 4
for step 6.
All solutions should be prepared in high quality deionised water
suitable for molecular biology.
1. Suspension Solution
50 mM Tris-HCl, pH 7.5 – 8.0, containing 10 mM EDTA and 100
‘micro’g/ml DNase-free RNase A.
Store at 4oC.
However, in the case of plasmid isolated from bacteria such as
Xanthomonas spp. or Pseudomonas spp., producing exo-polysaccharide
during culture, use either Suspension Solution containing 3 % NaCl or
just 3 % NaCl. Alternatively, approximately 3 % NaCl could be added
directly to a bacterial culture. Mix thoroughly before proceeding with
2. Lysis Solution
0.2 M NaOH containing 1 % SDS
3. Neutralising Solution
4 M potassium-acetate, pH 4.8
Place 23.55 g potassium acetate in measuring cylinder and fill to 66
ml mark with MQ water . Add 28.5 ml glacial acetic acid, mix and
titrate with about 1.5 ml of concentrated HCl to pH 4.8. Top up to 100
ml with MQ water .
4. Binding Buffer
6 M guanidine hydrochloride
It is not necessary for guanidine hydrochloride to be dissolved in TE
buffer as described in Hansen et al. as MQ water is equally good. 5 M
or 4 M works well but 6 M is preferable. Anything less than 3 M gives
5. Washing Solution
80% isopropanol . Ethanol is generally good as a washing solution,
except that isopropanol is cheaper.
6. TE Buffer
10 mM Tris-HCl, pH 8.5, containing 1 mM EDTA
7. Diatomaceous Earth Solution
The preparation of this solution is crucial. Suspend the diatomaceous
earth at 50 mg/ml in water and leave to sediment for more than 3 hrs.
Carefully discard as much of the water containing the white gelatinous
colloidal suspension as possible, but leave the sediment intact.
Repeat at least 3 times . If fine gelatinous matter is found during
use, then discard the supernatant carefully and replace it with the
same amount of water to maintain the same concentration of
diatomaceous earth as above. Again, any milky suspension of
diatomaceous earth should be removed as above. Even normal or low
quality diatomaceous earth gives much better yields than any of the
commercial kits tried. High quality diatomaceous earth is only
necessary when an ultrapure plasmid preparation is required. We have
not tested the difference between a highly pure plasrnid and an
ultrapure plasmid preparation, but we think that the results will be
the same as long as the plasmid purity is between 1.8 to 2.0
Preparation of the diatomaceous earth column
The diatomaceous earth solution should be resuspended thoroughly
Place a 2 – 5 ml syringe to a minicolumn and attach to a vacuum
fitting, but not apply vacuum as yet!
Load about 500 – 600 ‘micro’l of diatomaceous earth solution onto
the column and apply suction. Once all the solution has been applied,
watch the column from above and begin to apply gentle suction.
Disconnect the vacuum immediately when the liquid phase disappears and
the surface becomes solid. The column should look greyish white, with
a thin brilliant white band at the bottom. If the column is brilliant
white all the way up, the vacuum has been applied for too long. Dried
columns don’t bind DNA.
If you do not have a vacuum device or suitable setup, connect a
syringe to the top of the column via the luer lock and apply pressure
gently to obtain the same effect. Be sure to disconnect the syringe
from the column before pulling back on the plunger. The column is now
ready to be used in step 5 of the procedure section. The syringe can
be reused after cleaning with MQ water or distilled water. The column
can also be reused after appropriate cleaning as described below.
1) Remove the diatomaceous earth completely from the column.
2) Soak the column in 0.1 M HCl for at least 1 h and boil for 10 – 20
3) Wash it thoroughly using MQ water or distilled water and autoclave.
4) Fit a filter in the column using a yellow micropipet tip before
Key points to observe:
a. Use a endA1- E. coli strain for plasmid propagation and isolation
whenever possible. The instability of plasmids isolated from endA l +
bacterial strains has been reported .
b. Do not vortex, shake or incubate for more than 5 min in step 3.
This may cause shearing of genomic DNA and/or linerization of the
supercoiled plasmid. A Iysis time of less than 5 min is important to
cause maximum release of plasmid while minimising plasmid
denaturation. The lysate should be clear and viscous.
c. Use of cold room or less than 7 min centrifugation may give rise to
a dirty supernatant in step 4. If for whatever reason the
centrifugation has to be performed at low temperature, the mixture
should be transferred to room temperature as quickly as possible after
d. In earlier protocols and in protocols of commercial miniprep
plasmid purification kits, less than 1 min centrifugation is
recommended to remove ethanol from either the binding resin or a
diatomaceous earth column, but we found that under these conditions
some ethanol still remained in the diatomaceous earth. Therefore,
centrifugation should be at least 3 min in step 7. If necessary,
repeat the centrifugation twice. DNA will not be lost.
e. Use only half of the first volume during step 9. If 100 ‘micro’l is
used for the first elution, then we recommend less than 50 ‘micro’l
for the second elution. If the diatomaceous earth is found in the
bottom of the tube following centrifugation, transfer the supernatant
carefully into a new eppendorf tube.
Troubleshooting and Hints
Very low yields of plasmid – this is usually attributed to a loosely
fitting filter in the column. Check whether the filter in the column
is fitted lightly. Check the plasmid copy number. Was antibiotic added
Low purity of plasmid with an OD 260/280, greater or less than 1.8 –
2.0. This usually arises from white gelatinous matter remaining above
the diatomaceous earth when preparing the solution. Check the
diatomaceous earth solution. Check whether endA 1-/+ cells were used.
Vacuum is best applied from a steady source such as ‘house vacuum’.
Syringes tend to stick and give bursts of vacuum.
If the supernatant in step 5 contains cell debris in suspension
because of careless transfer, the column will clog. In this case do
not discard sample, but scratch column surface slightly with pipette
tip to unclog column.
Cheap ICN Practical Grade guanidine hydrochloride is quite suitable,
as long as undissolved solids are removed by filtration once the
theoretically 6M solution has been made up.
Home made filter columns can be made using microcentrifuge tubes as
described by Hansen et al. , but we recommend piercing the bottom of
the tube with a needle, from the inside, rather than snipping the
In principle it should be possible to scale this up to a macro-prep.
We have only worked with 20 ml cultures per prep.
Triton-Prep Method for bacterial DNA Purification
- Grow 5 . Harvest in single eppendorf tube .
- Resuspend pellet with 300ul STET buffer . After resuspending add
30ul RNase/lysozyme mixture .
- Boil for one minute 15 seconds .
- Spin in microfuge for at least 15 minutes.
- Take supernatant and phenol extract with 150ul STET- saturated
- Spin and take supernatant. Add 1/10 volume 4M lithium chloride .
Let sit on ice for 5-10 minutes.
- Spin and take supernatant. Add equal volume isopropanol. RT for 5
- Spin. No pellet will be visible. Don’t panic, DNA is stuck to side
all the way up tube.
- Important: Wash with 80% ethanol
- Resuspend pellet in 50-200ul.
Lysozyme/ RNase mixture
50mM Tris-HCl pH8.0
Store at -20oC in small aliquots. Do not refreeze after thawing.
5% Triton X-100
50mM EDTA pH 8.0
Filter sterilize. Store at 4oC