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
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 Bufferin 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.
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 (final concentration) could be added directly to a bacterial culture. Mix thoroughly before proceeding with step 2.
0.2 M NaOH containing 1 % SDS
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 .
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.
80% isopropanol (dilute to 80 % with MQ water). Ethanol is generally good as a washing solution, except that isopropanol is cheaper.
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.