可以在第一次酶切后将酶切样直接纯化

科研动态

核心提示:
做分子生物学实验,核酸纯化、酶切、克隆算是最基本的步骤了。因为要做定向克隆,通常要作双酶切。为了省时
做分子生物学实验,核酸纯化、酶切、克隆算是最基本的步骤了。因为要做定向克隆,通常要作双酶切。为了省时省事儿,我们常常想方设法把两个酶放在一起切。可是事情并非总那么称心如意——两个酶经常在一起闹别扭,不是反应温度不同啦,就是Buffer不同,有时候两个酶切位点连在一起,必须先切一个再切另一个,否则就切不动——因为有的酶除了识别位点之外还需要旁边留有几个碱基,如果正好被切掉了就出问题。于是有人就到处找通用Buffer——其实所谓通用Buffer也只勉强适用于少数几个常用酶,而且往往会出现不是最佳反应条件的问题、星号活力问题——想想看,酶在生物体内精密调控各种反应,不是最适条件当然容易出问题——马虎一下有时就会有莫名其妙的结果,该切的切不动,不该切的切掉了,该连接的连不上,倒霉上一次就能浪费你几个月时间。于是我们一股脑的怨:酶有问题。可是如果两个酶分别切吧,费时间不说,切完一个,还要纯化好半天——就是不跑电泳也得酚氯仿抽提,本来就不多的样品这么一折腾,够呛。幸好如今有了试剂盒,一切都简单了。您别说,这试剂盒虽然花钱,原理也简单,可是用起来就是方便。第一次酶切后,只要5、6分钟可以很方便地快速纯化酶切样,不用酚抽不用乙醇沉淀,就可以进行第二次酶切,保证最佳反应条件,又何必煞费苦心的作双酶切呢,还可以减少一些人为的错误了。

核心提示:IntroductionThis cheap and simplified protocol, based on
Hansen et al., 1995, gives high yields of plasmid DNA as w

MinElute
DNA纯化技术是基于一种特殊的、叫做Silica-gel-menbrane的膜,能够在高盐浓度的条件下迅速吸附DNA,在低盐或者纯水条件下释放DNA,只要短短几分钟,就能彻底除去包括引物、核苷酸、酶、矿物油、盐、琼脂糖、EB以及其它杂质,和以前QIAGEN的Silica-gel-menbrane有所不同的是,它要求的洗脱体积非常小,只要10微升,纯化的DNA是高度浓缩的,方便进行以后的反应。回收率在80%以上。

Introduction

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
typically obtained.

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.

Procedure

  1. Grow a 3 ml culture of E. coli overnight containing an appropriate
    antibiotic

  2. Harvest the cells by centrifugation for 2 min and suspend in 300 –
    500 ‘micro’l of Suspension Solution at room temperature.

  3. Add 300 – 500 ‘micro’l of Lysis Solution, mix very gently and keep
    at room temperature for about 5 min

  4. 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.

  5. 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.

  6. 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 .

  7. 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 .

  8. 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.

Solutions required:

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
step 2.

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
poor results.

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
before use.

  1. Place a 2 – 5 ml syringe to a minicolumn and attach to a vacuum
    fitting, but not apply vacuum as yet!

  2. 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
min.

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
use.

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
centrifugation.

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
or not?

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
bottom off.

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

  1. Grow 5 . Harvest in single eppendorf tube .
  2. Resuspend pellet with 300ul STET buffer . After resuspending add
    30ul RNase/lysozyme mixture .
  3. Boil for one minute 15 seconds .
  4. Spin in microfuge for at least 15 minutes.
  5. Take supernatant and phenol extract with 150ul STET- saturated
    phenol.
  6. Spin and take supernatant. Add 1/10 volume 4M lithium chloride .
    Let sit on ice for 5-10 minutes.
  7. Spin and take supernatant. Add equal volume isopropanol. RT for 5
    minutes.
  8. Spin. No pellet will be visible. Don’t panic, DNA is stuck to side
    all the way up tube.
  9. Important: Wash with 80% ethanol
  10. Resuspend pellet in 50-200ul.

Lysozyme/ RNase mixture

10mg/ml lysozyme

1mg/ml RNase

50mM Tris-HCl pH8.0

Store at -20oC in small aliquots. Do not refreeze after thawing.

STET

8% sucrose

5% Triton X-100

50mM Tris-HCl

50mM EDTA pH 8.0

Filter sterilize. Store at 4oC

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