Qiagen columns

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Wed Jun 8 11:27:05 EST 2011


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.
=3D=3DAlkaline Lysis=3D=3D
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 wit=
h
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. Incubat=
e
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 -20=
C
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:
=3D=3DBoiling Method=3D=3D
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, 5=
0
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. Wher=
e
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 =93fishing=94 it out with a sterile woo=
den
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 fo=
r
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 T=
E
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 <ebs15242 from creighton.edu> 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.
>
>
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> Methods mailing list
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>



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