CN1281765C - Method for preparing specimen in use for researching DNA through microscope in atomic force - Google Patents
Method for preparing specimen in use for researching DNA through microscope in atomic force Download PDFInfo
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- CN1281765C CN1281765C CN 200510016720 CN200510016720A CN1281765C CN 1281765 C CN1281765 C CN 1281765C CN 200510016720 CN200510016720 CN 200510016720 CN 200510016720 A CN200510016720 A CN 200510016720A CN 1281765 C CN1281765 C CN 1281765C
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- dna
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- mica
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Abstract
The present invention relates to a method for preparing a sample in use for researching DNA by an atomic force microscope. Firstly, 1 ng/mul of DNA and a proper amount of 1mMMg <2> of divalent metal ion mixed solution are heated at 37 to 40 DEG C for 30 to 60 minutes. 20 mul of solution is dripped on 1.5 cm2 of the surface of mica newly dissociated; the adsorption lasts for 5 to 8 minutes at 25DEG C; then, inorganic salt ions are diffused in ultra-pure water at 4 to 25 DEG C for 10 to 30 minutes. A sample is taken from water and put in anhydrous alcohol for 10 to 30 seconds; finally, the sample is dried for 2 hours at 25 DEG C under nitrogen protection. The DNA sample prepared by the diffusion method avoids the disadvantage of water flush, keeps the DNA molecule conformation and prepares the clean sample. The success ratio for preparing the sample reaches as high as more than 95%.
Description
Technical field
The invention belongs to and adopt diffusion process in the ultrapure water to prepare the method for sample, specifically a kind of preparation method of specimen in use for researching DNA through microscope in atomic force.
Background technology
Atomic force microscope is the opticmicroscope that continues, one of novel microscope of the third generation that grows up after the electron microscope.It relies on small probe to survey character such as the pattern of analyte and electricity thereof, magnetic, viscoelastic power, hardness.Owing to the resolving power of its superelevation, do not need substrate to have electroconductibility, advantage such as can operate under liquid and atmospheric environment, atomic force microscope has been widely used in the interaction of imaging DNA and researching DNA and other molecule.In these researchs, mica becomes widely used substrate with characteristics such as the single facet of its atomic-level flatness, super large and the new surface of preparation of dissociating easily.Therefore prepare the major objective that the DNA sample becomes people's research based on mica substrate.During employed in the past divalent-metal ion method fixed dna, be earlier DNA to be dropped in mica surface absorption several minutes (1 ~ 3 minute), then with the ultrapure water flushing, last dry air (as Nucleic Acids Research 24 (14) (1996) 713-720).This treatment process since in the process of water flushing uncontrollable property, make that the DNA sample success ratio of preparation is very low (destroyed that the part that is adsorbed onto suprabasil DNA structure picture and causes DNA is trooped etc.).Moreover the flushing of short period of time can not remove the salt ion that is adsorbed on the dna molecular effectively, makes to be that they separate out the imaging that the salifiable xln of shape has seriously disturbed DNA in the dry process on surface and dna molecular.A large amount of dna moleculars has also been removed in the water flushing in addition, makes the dna molecular that is adsorbed onto substrate surface become seldom, causes the waste of DNA sample.There are these problems based on the method for finishing in other too as silanization, Wu Er Chuoization etc.
Summary of the invention
In order to solve the defective that prior art exists, the theory that interacts and in the solution of two different concns, spread based on inorganic ion and DNA, improved water rinse step in the sample preparation in the past, the substrate that is adsorbed with the DNA sample has been placed on spreads preparation DNA sample in the ultrapure water.The preparation method who the purpose of this invention is to provide a kind of specimen in use for researching DNA through microscope in atomic force.
Step of the present invention is as follows:
1), with DNA and an amount of magnesium acetate mixing wiring solution-forming, the two basic proportioning is, 1ng/ μ lDNA:1mM magnesium acetate was in 37～40 ℃ of heating 30～60 minutes.Optimum temps is 38 ℃, and Best Times is 30 minutes;
2), with the above-mentioned mixing solutions of the DNA that heats, drop in new dissociated mica surface.Mica surface is 25 ℃ of room temperatures, adsorbs 5～8 minutes.Generally be that 20 μ l drop in 1.5cm
2On the surface;
3), the mica that will be adsorbed with dna solution is placed down in the ultrapure water (18.2M Ω cm) in 4～25 ℃ and spreads.The positively charged ion that is adsorbed on the DNA just spreads in ultrapure water, because positively charged ion loses, electronegativity increases and produces repulsive interaction and make dna molecular spontaneously disperse, launch on the DNA phosphoric acid skeleton;
4), then, take out sample and be placed in the dehydrated alcohol 10～40 seconds, the best is 30 seconds.The water molecules that is adsorbed on sample surfaces spreads in ethanol, and the drying of having quickened sample has also strengthened the stability of dna molecular.Drying at least 2 hours under 25 ℃, nitrogen protection condition at last.
The DNA sample of this method preparation characterizes through atomic force microscope and finds DNA dispersed better (two chains of dna molecular almost completely stretch and seldom have overlapping) and evenly (surface density of sample is even), sample cleaning (almost not having the xln of salt to be present on substrate surface and the dna molecular), sample repeatability can be up to more than 95%.Making processes is comparatively simple, practical, the DNA consumption is few.(condition is that DNA concentration is 10～25ng/ μ l, and dropping in the amount of mica surface and the basic proportioning of mica area is 20 μ l/1.5cm to can be used for preparing closely the dna single molecular film
2, be 10～30 minutes diffusion time), the linear DNA of full extension (condition be linear DNA concentration less than 2.5ng/ μ l, dropping in the amount of mica surface and the basic proportioning of mica area is 20 μ l/1.5cm
2, be 10～30 minutes diffusion time), the plasmid DNA of open loop (condition be plasmid DNA concentration less than 2.5ng/ μ l, dropping in the amount of mica surface and the basic proportioning of mica area is 20 μ l/1.5cm
2, 10～30 minutes diffusion times) etc.The sample of this method preparation is fit to the interaction of atomic force microscope imaging, particularly researching DNA molecule and other molecule very much.
The present invention has improved water rinse step in the sample preparation in the past, and the mica that is adsorbed with DNA is placed on the salt ion that spontaneous diffusion removal is adsorbed in the ultrapure water (18.2M Ω cm), makes the spontaneous expansion of dna molecular.Avoided the shortcoming of water flushing, the DNA sample success ratio of preparation is up to more than 95%.
Embodiment
Embodiment 1
12.5ng/ μ l λ～DNA and 12.5mM Mg
2+Mixing solutions is got its 20 μ l and is dropped in 1.5cm in 38 ℃ of heating 30 minutes
27～8 minutes (25 ℃) of new dissociated mica surface absorption were placed down in the ultrapure water diffusion 30 minutes with this mica in 4 ℃, were placed in the dehydrated alcohol 30 seconds after the taking-up, at last in 25 ℃, and nitrogen protection dry 2 hours down.Atomic force microscope characterizes this method and has prepared DNA unimolecular film closely.
Embodiment 2
2.5ng/ μ l plasmid DNA pBR 322 and 2.5mM magnesium acetate mixing solutions are got its 20 μ l and are dropped in 1.5cm in 38 ℃ of heating 30 minutes
27～8 minutes (25 ℃) of new dissociated mica surface absorption were placed down in the ultrapure water diffusion 30 minutes with this mica in 4 ℃, took out the back and placed 30 seconds in dehydrated alcohol, at last in 25 ℃, nitrogen protection dry 2 hours down.Atomic force microscope characterizes this method and has prepared the unfolded plasmid DNA molecule.
Embodiment 3
2.5ng/ μ l linear DNA pBR 322/Pst I and 2.5mM magnesium acetate mixing solutions, are got its 20 μ l and are dropped in 1.5cm after 30 minutes in 38 ℃ of heating
27～8 minutes (25 ℃) of new dissociated mica surface absorption were placed down in the ultrapure water diffusion 30 minutes with this mica in 4 ℃, were placed in the dehydrated alcohol 30 seconds after the taking-up, at last in 25 ℃, nitrogen protection dry 2 hours down.Atomic force microscope characterizes the linear DNA molecule that this method has prepared full extension.
Claims (3)
1, a kind of preparation method of specimen in use for researching DNA through microscope in atomic force is characterized in that:
1) with DNA and magnesium acetate mixing solutions, in 37～40 ℃ of heating 30～60 minutes, the basic proportioning of DNA and magnesium acetate was: 1ng/ μ l DNA:1mM magnesium acetate;
2) the above-mentioned mixing solutions of the DNA that heats being dropped in new dissociated mica surface adsorbed 5～8 minutes down in 25 ℃;
3) mica that will be adsorbed with dna solution spreads in 4～25 ℃ of ultrapure waters that are placed down in 18.2M Ω cm;
4) then, take out sample and be placed in the dehydrated alcohol after 10～40 seconds under 25 ℃, nitrogen protection condition drying at least 2 hours.
2, the preparation method of specimen in use for researching DNA through microscope in atomic force according to claim 1, when it is characterized in that described DNA concentration is 10～25ng/ μ l, the amount that DNA and magnesium acetate mixing solutions drop in mica surface is 20 μ l/1.5cm with the basic ratio of mica area
2, be to prepare the dna single molecular film in 10～30 minutes diffusion time.
3, the preparation method of specimen in use for researching DNA through microscope in atomic force according to claim 1, when it is characterized in that described DNA concentration greater than zero, less than 2.5ng/ μ l, the amount that DNA and magnesium acetate mixing solutions drop in mica surface is 20 μ l/1.5cm with the basic ratio of mica area
2, be to prepare the DNA of stretching, extension in 10～30 minutes diffusion time.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510016720 CN1281765C (en) | 2005-04-18 | 2005-04-18 | Method for preparing specimen in use for researching DNA through microscope in atomic force |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510016720 CN1281765C (en) | 2005-04-18 | 2005-04-18 | Method for preparing specimen in use for researching DNA through microscope in atomic force |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1696312A CN1696312A (en) | 2005-11-16 |
| CN1281765C true CN1281765C (en) | 2006-10-25 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510016720 Expired - Fee Related CN1281765C (en) | 2005-04-18 | 2005-04-18 | Method for preparing specimen in use for researching DNA through microscope in atomic force |
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| CN (1) | CN1281765C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101435759B (en) * | 2008-12-11 | 2012-06-27 | 上海交通大学 | Automatic DNA molecule operating method based on atomic force microscope |
| CN108680510B (en) * | 2018-04-24 | 2020-11-03 | 金华职业技术学院 | Surface nanostructure magnetic measurement method |
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2005
- 2005-04-18 CN CN 200510016720 patent/CN1281765C/en not_active Expired - Fee Related
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| Publication number | Publication date |
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| CN1696312A (en) | 2005-11-16 |
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Granted publication date: 20061025 |