Hi Ed, I can't speak authoritatively, but I'm pretty sure the columns are all the same material. Only the buffers should make a difference on what binds and what washes through. However, if it's for critical applications, don't try it out unless someone else chimes in to confirm what I've just said. :)
Don't forget minipreps are routinely done without columns too, they just require a few more spin cycles and a bit of care remembering which fraction contains the DNA at which step. Here are two quick protocols: First draft (can't find the final draft just now) of a "Standard" miniprep I wrote up to fit on a business card. Excuse brevity, and you can probably skip a lot of the incubations. ==Alkaline Lysis== All Centrifugation is performed at 8Krcf-10Krcf. Use Deionised H20 for everything. Buffer I: 0.2g Lysozyme, 0.9g Glucose, 0.3g EDTA, 0.3g Tris to 100ml with H20 Buffer II: 0.8g NaOH, 1ml SDS, to 100ml with H20 Buffer III: 24.6g NaAc, Adjust to pH4.8 with Acetic Acid, to 100ml with H20 Buffer TE: 1.21g Tris, 0.3g EDTA, Adjust to pH8 with Acetic Acid, to 1L with H20 Step 1: Isolate cells from 0.5ml broth, grown overnight, by centrifugation for 1m Step 2: Remove liquid phase, resuspend cell pellet in 0.1ml Buffer I. E.coli: Incubate at 0C for 30 minutes. B.subtilis: Incubate at 37C for 30 minutes. Step 3: Add 0.2ml of Buffer 2, cap & invert x10 to mix thoroughly. Incubate 0C for 5m. Step 4: Add 0.15ml of Buffer 3, cap & invert x10 to mix thoroughly. Incubate 0C for 60m. Step 5: Centrifuge for 10m. Carefully remove 0.4ml of clear liquid phase to new tube. Step 6: Discard solids. To liquid, add 2.25ml 70% Isopropanol. Incubate -20C for 20m. Step 7: Centrifuge for 10m. Carefully remove liquid, leave clear/white DNA pellet. Step 8: Resuspend in 0.1ml Buffer NaTH, add 0.2ml Ethanol/Isopropanol. Incubate at RT for 30m. Step 9: Centrifuge for 10m. Carefully remove liquid, leave clear/white DNA pellet. Step 9a: Optionally re-precipitate DNA with Ethanol/Isopropanol again. Then air-dry fully. Step 10: Resuspend in Buffer TE carefully. If completely dry this may take some time. Alternative method using boiling that I found a few days ago on a blog: ==Boiling Method== An alternative to alkaline lysis method that was developed by Holmes and Quigley. Here, the cells are lysed partially allowing plasmids to escape, whereas the bacterial chromosomal DNA remains trapped in the cell debris. High temperature is then used to denature the chromosomal DNA, after which reannealing allows the plasmids to reassociate. Centrifugation removes the chromosomal DNA along with the cell debris, leaving the plasmid in suspension, from where it is recovered by isopropanol precipitation. Materials: LB broth bacteria culture medium. The content are 1% Tryptone, 0.5% yeast extract, 200 mM NaCl, then it sterilize by autoclaving in suitable aliquots. STET mix which is contain 5% (v/v) Triton X-I 00, 50 mM Tris-HCl, pH 8.0, 50 mM EDTA, pH 8.0, 8% (w/v) sucrose. Store the mix solution at room temperature. Lysozyme: Dry powder. Store at -20oC. 70% Ethanol. Isopropanol. TE solution: 10 mM Tris-HCl pH 8.0,1 mM EDTA. A boiling water bath: An opened bottom tube rack is required because the tubes must be placed directly in the water to achieve rapid heating. Sterile wooden toothpicks. Methods: Set up a culture for each miniprep by inoculating 2-3 mL of L-broth, containing an appropriate antibiotic (e.g., 100 micrograms/mL ampicillin) with a bacterial colony. Grow overnight at 37oC with vigorous shaking. Where plasmids have a high copy number, the growth time may be reduced to approx 6 h. Before starting the miniprep, begin boiling the water and make up a fresh solution of 1 mg/mL lysozyme in STET mix. Fill a 1.5-mL labeled microfuge tube with an aliquot from each culture. Pellet the bacteria by centrifugation for 1 min at 12,000 g. Carefully aspirate off the supernatant using a drawn-out Pasteur pipet. The short centrifugation time leaves a loose pellet that is easier to resuspend. If the pellet does not readily resuspend, pipet the solution up and down to dislodge it. Do not suck the pellet directly into the pipet tip. Vortex each pellet for a few seconds to break up the pellet. Add 20 micrliters STET mix to each tube. The pellet should now easily resuspend by vortexing. Immediately place the tubes in the open-bottom rack, and place in the boiling water for exactly 45 s. Ensure that each tube is at least half submerged. Centrifuge the tubes at 12,000 g for 10 min. A large, sticky, loose pellet should form. Remove the pellet from each tube by “fishing” it out with a sterile wooden toothpick. Because the pellet is quite slippery, it is useful to have a paper tissue at the top of the tube to catch the pellet and prevent it from slipping back down into the tube. Add 200 microliters isopropanol to each tube, and centrifuge at 12,000 g for 5 min. Aspirate the supernatant, and wash the pellet in 500 microliters 70% ethanol. Centrifuge the tube for 1 min to compact the pellet, and then aspirate the 70% ethanol. Air dry the pellets for 10 min, and resuspend each one in 100 microliters TE buffer. Vortex and shake for 10 min before use to ensure complete dissolution. Use 10 microliters (equivalent to 100 ng of plasmid for most vectors) and analyze by gel electrophoresis. All the best, Cathal On 8 June 2011 16:42, Ed Siefker <[email protected]> wrote: > I'm a little short on mini-prep columns today. > Can I use RNEasy to substitute? I've used > the pink PCR purification columns in the past > with success, but I'm not sure if the RENasy > columns are similar. > > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > -- letters.cunningprojects.com twitter.com/onetruecathal http://www.indiebiotech.com _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
