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Purification of plasmid DNA (miniprep) with high yields using diatomaceous earth

<center> <font>Kyung-Soo Kim and Charles K. Pallaghy</font> </center>

 

<center> <font> <font> School of Botany, La Trobe University, Bundoora Vic 3083, Australia <br /> <br /> </font> </font> </center>

Correspondence to C. K. Pallaghy, e-mail C.Pallaghy@latrobe.edu.au

Copyright K-S. Kim and C. K. Pallaghy; 1996, All rights reserved. Modified 3/10/97

 

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 (Sambrook et al. , 1989). 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 (e.g. pGEM-3Zf(+), Promega, Madison, WI, USA) can be obtained at a purity of 1.8 to 2.0 (260/280). 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 (Birnboim, 1983) and elution of DNA from a home-made diatomaceous earth binding matrix (Hansen et al., 1995). We used a Promega WizardTM minicolumn (Madison, USA), but other brands/types of columns can also be used. Centrifugation in the protocol is carried out at 13,000 rpm (or at least > 10, 000 rpm) on a minifuge unless otherwise specified.

Procedure

1. Grow a 3 ml culture of E. coli overnight (at least 16 hrs) containing an appropriate antibiotic (e.g. ampicillin 25-50 'micro'g/ml)

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 (but no more than 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 (see instructions for preparation of columns as mentioned later). Although diatomaceous solutions store well, the columns don't.

5. Carefully transfer the supernatant and mix with approximately the same volume of Binding Buffer in a syringe and apply the mixture to the top of a freshly made diatomaceous earth column (see instruction for preparation of columns as mentioned later). 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 (to obtain high quality plasmids).

7. Place the washed and drained column into a new eppendorf tube and add 50 'micro'l of preheated MQ water (70 - 80 °C) or TE buffer (at room temperature) to elute the DNA and place at room temperature for 10 min (but no more than 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 (MQ) 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 4 ^o C.

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 (final concentration) 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. (1995) 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 (dilute to 80 % with MQ water). 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 (Sigma D-5384 or other brands) 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 (the more, the better). 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 (but acid-washed) 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 (OD 260/280)

 

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 (25 - 30 mg) 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 (Schoenfield et al. , 1995).

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 (or unravelling) 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

(i) 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?

(ii) 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.

(iii) Vacuum is best applied from a steady source such as 'house vacuum'. Syringes tend to stick and give bursts of vacuum.

(iv) 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.

(v) 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.

(vi) Home made filter columns can be made using microcentrifuge tubes as described by Hansen et al. (1995), but we recommend piercing the bottom of the tube with a needle, from the inside, rather than snipping the bottom off.

(vii) In principle it should be possible to scale this up to a macro-prep. We have only worked with 20 ml cultures per prep.

 

References:

1. Birnboim, H. C. 1983. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 100 , 243 - 255.

2. Hansen, Nils Jakob V., P. Kristensen, J. Lykke, K. K. Mortensen and B. F. C. Clark. 1995. A fast, economical and efficient method for DNA purification by use of a homemade beads column. Biochemistry and Molecular Biology Intemational 35 (3) , 461 - 465.

3. Sambrook J. et al., 1989. Molecular Cloning; A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

4. Schoenfeld, T., J. Mendez, D. R. Storts, E. Portman, B. Patterson, J. Frederiksen and C. Smith. 1995. Effects of bacterial strains carrying the endAI genotype on DNA quality isolated with Wizard Plasmid Purification System. Promega Notes , 53 , 12 - 22.

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Triton-Prep Method for bacterial DNA Purification

1. Grow 5 ( large scale-15ml culture). Harvest in single eppendorf tube (or 15 ml disposable tube).

    

2. Resuspend pellet with 300ul STET buffer (900ul). After resuspending add 30ul RNase/lysozyme mixture (100ul).

    

3. Boil for one minute 15 seconds (one minute 45 seconds).

    

4. Spin in microfuge for at least 15 minutes.

    

5. Take supernatant and phenol extract with 150ul (500ul) STET- saturated phenol.

    

6. Spin and take supernatant. Add 1/10 volume 4M lithium chloride (autoclaved). 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 (95% will cause the residual Triton to precipitate)

    

10. Resuspend pellet in 50-200ul.

     

STET

Lysozyme/ RNase mixture

10mg/ml lysozyme

1mg/ml RNase (use cheap grade (BMB) rather than RNase A , which is too expensive)

50mM Tris-HCl pH8.0

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

8% sucrose

5% Triton X-100

50mM Tris-HCl (pH8.0)

50mM EDTA pH 8.0

Filter sterilize. Store at 4oC

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Bacterial Genomic DNA Purification via Qiagen columns

Cultures should be grown in LB for best results. To avoid overloading the Qiagen columns, the correct number of cells/volume of culture should be determined. Qiagen recommends not more than 4x10E9 cells for a mini-column, 2x10E10 for the midi, and 8x10E10 for the maxi. For this is ~0.4-0.8 ml, 2.0-4.0 ml, and 10.0 to 20.0 ml for overnight cultures in LB.

1. Prepare buffers according to recipe at end. Equilibrate all to room temp. before use.

 

			Amount/prep   Mini  Midi  Maxi
			B1    1 ml  3.5 ml  11 ml
			B2    0.35 ml  1.2 ml  4 ml
			QBT    2 ml  4 ml  10 ml
			QC    3 ml  15 ml  30 ml
			QF    2 ml  5 ml  15 ml
			RNaseA    0.2 mg  0.7 mg  2.2 mg
			lysozyme   2 mg  8 mg  30 mg
			Qiagen proteasundefined  0.9 mg  2 mg  10 mg
		~undefinedor proteinase K
		

2. Dissolve Rnase A in buffer B1 to concentration of 200 ug/ml. Stock solutions of lysozyme and proteinase K can be made in dH2O to concentrations of 100 mg/ml and 20 mg/ml respectively.

3. Pellet cells by centrifugation at 3000-5000x g for 5-10 min. Remove supernatant.

4. Resuspend the bacterial pellet in 1/3.5/11 ml (mini/midi/maxi columns) of Buffer B1 by vortexing at top speed.

5. Add 20/80/300 ul of lysozyme stock solution and 45/100/500 ul of proteinase K. Incubate at 37 C for at least 30 min.

6. Add 0.35/1.2/4 ml of Buffer B2 and mix by inversion several times. Incubate at 50 C for 30 min. Mix well; this step is important for efficient deproteinization. It is also important that the lysate becomes clear at this stage.

7. Equilibrate Qiagen genomic tip with 2x1/4/10 ml Buffer QBT, and allow the tip to empty by gravity flow.

8. Vortex the lysate for 5-10 sec and apply onto the equilibrated column. Again, allow it to pass through column by gravity flow. Flow can be assisted with gentle positive pressure (a plunger from a 10 ml syringe fits the midi columns) but it is also OK to dilute the lysate with an equal volume of Buffer QBT prior to loading. The latter is preferable by virtue of personal experience.

9. Wash the column with 3x1 ml/2x7.5ml/2x15 ml of Buffer QC. Allow buffer to pass through by gravity flow. Two washes should be enough.

10. Elute the genomic DNA with 2x1 ml/5 ml/15 ml of Buffer QF. Precipitate with 0.7 volumes (1.4/3.5/10.5 ml) isopropanol, equilibrated to room temperature. The DNA should be spoolable; if not, pellet the precipitate by centrifugation at 5000+ x g. Wash the precipitate with 70% EtOH and dry. Resuspend in the appropriate solvent (TE, water).

 

			Solutions:
			Buffer  Composition (Storage)
			B1 50 mM EDTA, 50 mM Tris/HCl, 0.5% Tween 20, 0.5%
			Triton X-100 (room [4 C after addition of RNase])
			B2 3 M GuHCl, 20% Tween 20 (room temp)
			QBT 750 mM NaCl, 50 mM MOPS, 15 % ethanol, 0.15% triton
			X-100, pH 7.0 (room temp.)
			QC 1.0 M NaCl, 50 mM MOPS,  15% ethanol, pH 7.0 (room temp.)
			QF 1.25 M NaCl, 50 mM Tris/HCl, 15 % ethanol, pH 8.5 (room temp.)
		

Chromosomal DNA Extraction from Gram-positive Bacteria
This procedure was originally developed for Listeria monocytogenes but has worked well with other Gram+ bacteria we've tried.

Pellet cells from 10 ml overnight cultures in BHI or LB and wash in 5 ml of 0.1X SSC.
Resuspend in 1 ml 10 mM Tris-HCl (pH 8.0) containing 20 % sucrose (v/v), add lysozyme to 2.5 mg/ml, and incubate at 37 C for 45 min.
Add 9 ml lysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 500 mg pronase B, 1 % SDS), and incubate additional 30 min at 37 C.
Phenol and chloroform extract lysed cells, and ethanol precipitate the DNA with 0.1 vol. 3 M sodium acetate, pH 4.8 and 2 vol. 95% ethanol.
Spool out DNA with a glass rod, wash once with 80% ethanol before drying.
Some bacterial species may require a longer incubation in lysozyme. For Renibacterium salmoninarum (a G+ salmon pathogen we work with), lysozyme incubations overnight at 37 C worked very well with high yields of DNA

ref: Flamm, R. K., Hinrichs, D. J., and Thomashow, M. F. 1984. Introduction of pAMb1 into Listeria monocytogenes by conjugation and homology between native L. monocytogenes plasmids. Infect. Immun. 44:157-161. 
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