Abbreviated miniprep protocol... Incredible!
pgegen at rnaworld.bio.ukans.edu
pgegen at rnaworld.bio.ukans.edu
Fri Mar 1 20:01:29 EST 1996
In <marcusj-2902961430440001 at path-myerson.mgh.harvard.edu>, marcusj at helix.mgh.harvard.edu (Jeremy Marcus) writes:
>The following miniprep protocol leaves solutions I, II, and III in the dust.
>I just recently discovered it in BioTechniques; here's the reference:
>Goode, BL and SC Fienstein. 1992. "Speedprep" purification of template for
>double-stranded DNA sequencing. BioTechniques 12:374-375.
>Actually this is a slight modification of the original:
>He, M., A. Wilde and MA Kaderbhai. 1990. A simple single-step procedure
>for small-scale preparation of Escherichia coli plasids. Nucleic Acids
>but I haven't checked that one out yet. The more recent one is all you need.
>If you can't get your hands on the article, here's what you need to know:
>1) Spin down 1.5 ml culture 30 sec
>2) Aspirate sup.
>3) Resusp pellet in 100 ul solution A (see end for recipe)
>4) Add 100 ul phenol/chloroform, vortex 10 sec
>5) Spin 2 min
>6) Save top layer, discard bottom
>7) Add 200 ul cold 100% EtOH, vortex briefly
>8) Spin 5'
>9) Wash with 1 ml 70% EtOH
>10) Speed-vac under low heat until dry (~2 min)
>11) Resusp in 10 ul TE with 100 ug/ml RNase A
> (I have found that I need to use actually a little more RNase than this.)
>12) Leave at room temp 5 min
>Solution A is:
>50 mM Tris pH 8.0
>4% Triton X-100
>2.5 M LiCl
>62.5 mM EDTA
>That's it, folks, you'll have 3 ug DNA in that 10 ul volume by the time
>you're through. Saves tons of time and the DNA is ready for restriction
>analysis (I have tried this) and even sequencing (I have not tried this,
>but they claim clean sequence the same quality as from cesium
>I'm going to pour my I, II, and III down the drain!
>QUESTION: Does anyone out there who already uses this procedure know how
>long one can use a preparation of Solution A before it goes bad?
??? Why is this exciting? Purification of RNA (and low-mol-wt DNA) by phenol extraction of bacterial cells is a technique that's at least 20 years old. The method posted seems pretty reasonable. I've used a similar protocol (perhaps the NAR version) and it worked quite well.
The Solution A will have a virtually indefinite shelf-life at room temperature; the only labile component is the Trition X-100 which may become oxidized over time.
ADDITIONAL INFO: 1) in this prep you may be able to reduce or avoid the use of RNase (for RE digests, certainly) merely by precipitating the RNA before the DNA: before adding EtOH, let the solution stand on ice 20 min. Spin down the precipitate of high-mol-wt RNA (mRNA & rRNA), remove the supernatant, and procede to precipitate the DNA.
(High-mol-wt RNA (mRNA & rRNA) precipitates at 2-2.5 M NH4OAc or 2.5-3 M LiCl, whereas tRNA, originally called 'sRNA' for 'soluble RNA', does not.)
2) If you need to prepare high-quality plasmid without the use of RNase (e.g., for use as an RNA polymerase template), the most effective RNA precipitant is 0.1 M MgCl2. A mini-prep based on this is given by Yamamoto & Horikoshi, NAR 23, 3351-3352 (1995). This proptocol also uses the old-time trick of precipitating DNA with CTABr detergent. This protocol does give sequenceable DNA.
3) Phenol extraction of whole E. coi cells leaves chromosomal DNA trapped inside the sacculus (the bacterial cell wall, a single cross-linked peptidylglycan molecule). Other procedures will release linear fragments of chromosomal DNA. These are most easily removed by extraction with phenol equilibrated to pH 4.8 - 5.0; the DNA partitions into the phenol phase.
When in doubt, simplify!
| Dr. Peter Gegenheimer | voice: 913-864-3939 FAX:913-864-5321 |
| Departments of Biochemistry | pgegen at kuhub.cc.ukans.edu |
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