Matthew Lyon – Rooselab - GeneChip Protocol for DNA hybridizations derived from Borevitz’s Arabidopsis protocol
Claire Federici modified and used non-nucleon'ed version March 19-21, 2007
rewritten Mon Jul 23 10:51:06 PDT 2007 to include Nucleon resin steps.
FROM FRESH LEAF MATERIAL STARTING POINT
Wed Sep 26 17:52:30 PDT 2007 An Alternative Procedure, using the Qiagen Kit w/o Ammersham
Kit (lessons learned from Ammersham kit): Organic Solvent Washing of Plant Tissue Before ‘Qiagen
DNeasy Plant’ to Try and Achieve Cleaner DNA
I noticed a few months ago that heptane, alcohol, detergent mixes completely (chemically) dissolve Citrus
plant tissue. It yields a polar DNA containing mass of plant tissue soaked with alcohol and some detergent
and solvent. I even tried filtering this through paper to try and get the DNA out but didn’t really find
anything quick’and’easy. The DNA stayed stuck to the polar plant material and filter paper. Not knowing
what to do with this vegetable mass (mess) I’d made  I gave up.
Then, a few months later, I revisited this step of the procedure, integrating it with the laboratory standard
Qiagen DNeasy procedure. The operating principles guiding my experimentation were ‘something in
Citrus DNA extractions are killing our ability to work with it’, ‘solvent wash steps remove (float away) bad
crap from my precious DNA samples’ and ‘the more of this solvent, the cleaner it will be’.
REQUIRED:
- liquid nitrogen
- heat block at 65 degC
- heptane:alcohol 50/50 (v/v) mixture (use HPLC grade n(normal)-heptane = 1-heptane. Do not use
hexane or mixed hexanes because of the Parkinson’s disease potential.)
- AP1 Qiagen’s EDTA and SDS lysing buffer
- AP2 Qiagen’s acetic acid mixture - the acetic acid helps precipitate polysaccharides and detergents in
the procedure, also, DNA binding to silica (the second, white column) occurs better at low pH.
- AP3/E Qiagen’s binding buffer must be some sort of alcoholic chaotropic salt (guandinium) force-thedna-to-the-column mix. The E may denote ‘elution’ or ‘EDTA’ perhaps. Who knows/cares.
- AW Qiagen’s wash buffer – an alcoholic (quandinium?) wash
- 90-95-100% alcohol as final (white) column as a washing and drying agent
- ddH2O for elution of your sample
- RNAse A from the kit (throw 5uL into some of the above solutions if you want.)
This protocol consists of a solvent-based washing the plant tissue before starting the standard Qiagen
protocol. Then, the standard Qiagen protocol is then modified by putting the AP2 detergent precipitation
step after the QIAshredder (filter) column - - this is so that DNA is not precipitated to adhere to the plant
debris and that detergents are precipitated from a relative clear solution after the QIAshredder column.
How the initial step is designed to work: at room temperature: this procedure puts the plant tissue
immediately into a buffer with alcohol, EDTA, and SDS present; where heptane floats on top. The SDS
and EDTA in the aqueous layer should inactivate any released water-soluble-enzymes capable of
degrading or modifying any DNA present. This initial plant tissue and solvents mixture is then vortexed to
form a brief emulsion. Separation quickly occurs, forming a biphasic organic/aqueous extraction of lipid
soluble molecules. The alcohol in the lower layer helps dissolve cellulose containing materials in the
presence of detergent and a non-polar solvent. (The mechanism of this may be the heptane-assisted
removal of water/weather/UV-proofing compounds protecting the cell wall and its precious contents,
especially true in the cuticle. Many of these compounds have interesting properties, like self-polymerization
under certain conditions. Self-polymerization to the DNA you’re trying to extract could be disastrous.)
Further, the alcohol seems to do something with hydrogen bonding disruption of plant cell walls. Who
knows/cares.).
Some of the organic nonpolar solvent may also help denature enzymes by haphazardly/fluidly associating
with any hydrophobic protein regions. The alcohol should also help mitigate some enzymatic reactions
and helps keep DNA ‘pushed’ (adsorbed) to the remaining cellulose-containing plant debris matrix when
centrifuged.
Page 1 of 11
Matthew Lyon – Rooselab - GeneChip Protocol for DNA hybridizations derived from Borevitz’s Arabidopsis protocol
Claire Federici modified and used non-nucleon'ed version March 19-21, 2007
rewritten Mon Jul 23 10:51:06 PDT 2007 to include Nucleon resin steps.
After centrifugation, the liquid AP1/alcohol and heptane layers are discarded and the result is a pellet of
only half-lysed (it hasn’t been heated yet) plant material containing a trace of heptane, some alcohol, some
AP1 buffer, and the DNA of interest. The resulting plant debris pellet finally suspended into plain AP1
buffer, further diluting any traces of the added alcohol and heptane.
I suspect that many heptane- and alcohol- soluble compounds from the Citrus leaf tissue are removed
through these steps. This is important for two reasons: 1) these compounds may react with the DNA of
interest (a lot of enzymes and substrates in plant cells undergo magnificent re-dox chemisty. This tries to
put a damper on that ) and 2) these compounds may interfere with ingredients of the Qiagen kit. An
example of this second type of interference is that the nonpolar solvent and lipid-associated molecules can
prevent polar molecules (like DNA) from sticking to second, white Qiagen column (the silica DNA-binding
one), or that the amount of detergent supplied in the AP1 buffer is perhaps insufficient to deal with the
quantity or type of nonpolar or semi-polar chemical compounds present in Citrus leaves. The solvent
extraction may also be removing components of the Qiagen kit (tested for, looks minimal) that somehow
affects (for the better?) the extraction process, as well.
--THE PROCEDURE:
Wash the collected leaves (young or mature) with a SDS and ddH2O rinse to remove any surface dirt,
pathogens, insects, etc… This is easily accomplished by adding a touch of SDS to the plastic collection
bags and using the deionized water faucet stream to form a detergent solution and then wash away any
contaminants. In doing this it is observed that mud and dirt do go down the drain. The resulting leaf tissue
also appears to lose a bit of their waxy appearance after this step, which may be indicative of some
epithelial waxes being removed by this step. The tissue is then to be frozen in the -80°C freezer until readyto-use.
Prepare a microfuge tube of AP1 buffer (400uL) and 400 uL of a 50:50 heptane:ethanol mix. The presence
of AP1 (an aqueous solution of SDS and EDTA) will suck the ethanol layer out of the heptane:ethanol
mix. Verify this occurs to verify that heptane is indeed present on top of the mix. Try to avoid using
substitute solvents for the heptane, hexane is considerably more volatile and toxic (causes irreversible
neurological damage by both demyelization of nerve tissue and generation of a specific neurotoxin in
vivo.), and chloroform is more dense than water. This plant tissue pre-wash procedure is dependent on the
nonpolar phase being less dense than water. Further, chloroform is capable of eating the plastic utilized in
many microfuge tubes, especially when centrifuged (it also eats pipettemen gears, evidently.)
Grind your tissue with at least three applications of liquid nitrogen and grinding to ensure a fine power.
Add enough spatulas scoops of ground tissue to the heptane:AP1/ethanol solultion tube. Do not add more
than the contents of the tube are still liquid. Vortex this vile chemical mix a bit and then spin it at maximum
velocity in the centrifuge at maximum velocity. Carefully remove it from the centrifuge and observe the
contents. It will be observed that the heptane layer accumulates chlorophylls and other lipids. The
chlorophylls occupy the liquid space and some lipid material pellets to the side of the tube near the
heptane phase. The plant tissue pellets to the bottom of the tube and has a somewhat colored and alcoholic
AP1 supernatant over it.
Discard all supernatant(s)
keep the pelleted plant tissue.
To the pelleted plant tissue add 400 uL AP1.
7. Add 400 μl Buffer AP1 and 4 μl RNase A stock solution (100 mg/ml) to a
maximum of 100 mg (wet weight) or 20 mg (dried) disrupted plant or fungal
Page 2 of 11
Matthew Lyon – Rooselab - GeneChip Protocol for DNA hybridizations derived from Borevitz’s Arabidopsis protocol
Claire Federici modified and used non-nucleon'ed version March 19-21, 2007
rewritten Mon Jul 23 10:51:06 PDT 2007 to include Nucleon resin steps.
tissue and vortex vigorously. No tissue clumps should be visible. Vortex or
pipet further to remove any clumps. Clumps of tissue will not lyse properly and
will therefore result in a lower yield of DNA. In rare cases, where clumps cannot
be removed by pipetting and vortexing, a disposable micropestle may be used.
Note: Do not mix Buffer AP1 and RNase A before use.
Vortex.
Heat to 65 degC for 10 minutes with occasionally sample mixing. Do not heat above 65 degC as this would
denature the DNA we’re attempting to extract. Note the color change of the lysate (from greenish to
brownish) – this is normal.
8. Incubate the mixture for 10 min at 65°C. Mix 2 or 3 times during
incubation by inverting tube. This step lyses the cells.
WITHOUT DOING THE 130uL of AP2 ADDITION, apply this lysate (500 uL worth) to the QIAshredder
column (the PURPLE one).
Ignore all the crap about spinning the lysate for plants with viscous lysates, etc. Spin at maximum velocity
for two minutes. This may form a greenish pellet of some plant debris at the bottom of the tube but we’re
going to resuspend it and get it to play with AP2 before we try to pellet all of the detergents and
polysaccharides out of solution. Notice we have not used ice to force precipitation in this procedure. We’re
going to take advantage of the polysaccharides in Citrus that have actually dissolved into the AP1
detergent solution to create a precipitate of both polysaccharides and detergents.
To do this with the purple column, to the flow-through add (in the collection tube) the 130 uL of AP2 (an
acetic acid solution). Replace the purple column section to ‘seal’ the tube a bit and vortex to resuspend any
pellet from the previous centrifugation.
10. Recommended: Centrifuge the lysate for 5 min at 20,000 x g (14,000
rpm). Some plant materials can generate very viscous lysates and large
amounts of precipitates during this step. This can result in shearing of the DNA in
the next step (see “Lysate filtration with QIAshredder”, page 21). In this case,
optimal results are obtained if the majority of these precipitates are removed by
centrifugation for 5 min at 20,000 x g (14,000 rpm). After centrifugation, apply
supernatant to QIAshredder Mini spin column and continue with step 11.
11. Pipet the lysate into the QIAshredder Mini spin column (lilac) placed in
a 2 ml collection tube, and centrifuge for 2 min at 20,000 x g (14,000 rpm). It
may be necessary to cut the end off the pipet tip to apply the lysate to the
QIAshredder Mini spin column. The QIAshredder Mini spin column removes most
precipitates and cell debris, but a small amount will pass through and form a
pellet in the collection tube. Be careful not to disturb this pellet in step 12.
Notice 9 is pasted here out-of-order: 9. Add 130 μl Buffer AP2 to the lysate, mix,
and incubate for 5 min on ice. This step precipitates detergent, proteins, and
polysaccharides.
Put the purple column and collection tube back into the centrifuge and centrifuge at maximum speed for
two minutes again. Carefully remove it from the centrifuge and note that a pellet has formed. The pellet
usually looks like a colony of Aspergillus on bread or a colony-plate.
Carefully pour off the supernatant to keep in a new tube and then add 675 uL of the AP3/E binding buffer.
Page 3 of 11
Matthew Lyon – Rooselab - GeneChip Protocol for DNA hybridizations derived from Borevitz’s Arabidopsis protocol
Claire Federici modified and used non-nucleon'ed version March 19-21, 2007
rewritten Mon Jul 23 10:51:06 PDT 2007 to include Nucleon resin steps.
13. Add 1.5 volumes of Buffer AP3/E to the cleared lysate, and mix by
pipetting. For example, to 450 μl lysate, add 675 μl Buffer AP3/E. Reduce the
amount of Buffer AP3/E accordingly if the volume of lysate is smaller. A
precipitate may form after the addition of Buffer AP3/E, but this will not affect the
DNeasy procedure. Note: Ensure that ethanol has been added to Buffer AP3/E.
See “Things to do before starting”, page 24. Note: It is important to pipet Buffer
AP3/E directly onto the cleared lysate and to
mix immediately.
Transfer 500 uL of this to the QiaSpin mini column (a silica-based DNA binding column). I usually apply
the binding buffer mix twice to the white silica column. Make sure the centrifuge is not set at max speed
anymore for this white spin column stuff.
14. Pipet 650 μl of the mixture from step 13, including any precipitate that
may have formed, into the DNeasy Mini spin column placed in a 2 ml
collection tube (supplied). Centrifuge for 1 min at _6000 x g (corresponds to
_8000 rpm for most microcentrifuges), and discard the flow-through.*
Reuse the collection tube in step 15. 15. Repeat step 14 with remaining
sample. Discard flow-through* and collection tube.
two
DO the
AW washes. Perhaps split the AW into a container labeled AW1 and AW2 so that you do
‘both’ and your brain remembers you did ‘both’.
DO the extra alcohol wash (100% ethanol won’t elute your DNA at all.)
16. Place the DNeasy Mini spin column into a new 2 ml collection tube
(supplied), add 500 μl Buffer AW, and centrifuge for 1 min at _6000 x g
(_8000 rpm). Discard the flow-through and reuse the collection tube in step
17. Note: Ensure that ethanol is added to Buffer AW. See “Things to do before
starting”, page 24. 17. Add 500 μl Buffer AW to the DNeasy Mini spin column,
and centrifuge for 2 min at 20,000 x g (14,000 rpm) to dry the membrane. It
is important to dry the membrane of the DNeasy Mini spin column since residual
ethanol may interfere with subsequent reactions. This centrifugation step ensures
that no residual ethanol will be carried over during elution. Discard flow-through
and collection tube. After washing with Buffer AW, the DNeasy Mini spin column
membrane is usually only slightly colored. In the rare case that the membrane
remains significantly colored after washing with Buffer AW, refer to “Darkly
colored membrane or green/yellow eluate after washing with Buffer AW” in the
Troubleshooting Guide on page 45. Note: Following the centrifugation, remove
the DNeasy Mini spin column from the collection tube carefully so the column
does not come into contact with the flowthrough, as this will result in carryover of
ethanol.
Elute into NO MORE THAN 100 uL of ddH2O (re-elute back through to get []’s up – this works by prewetting the column with the water, whereas the second elution carries the softened/release DNA out more
efficiently) or 500 uL of ddH2O, followed by heat volatilization of the extra ddH2O only if your endpoint
can handle ssDNA (note this makes it impossible to quantify or characterize your end product, so only do
this if you’re really desperate to get as much DNA off the silica column as possible.)
18. Transfer the DNeasy Mini spin column to a 1.5 ml or 2 ml
microcentrifuge tube (not supplied), and pipet 100 μl Buffer AE directly
onto the DNeasy membrane. Incubate for 5 min at room temperature (15–
25°C), and then centrifuge for 1 min at _6000 x g (_8000 rpm) to elute.
Elution with 50 μl (instead of 100 μl) increases the final DNA concentration in the
Page 4 of 11
Matthew Lyon – Rooselab - GeneChip Protocol for DNA hybridizations derived from Borevitz’s Arabidopsis protocol
Claire Federici modified and used non-nucleon'ed version March 19-21, 2007
rewritten Mon Jul 23 10:51:06 PDT 2007 to include Nucleon resin steps.
eluate significantly, but also reduces overall DNA yield. If larger amounts of DNA
(>20 μg) are loaded, eluting with 200 μl (instead of 100 μl) increases yield. See
“Elution”, page 21. 19. Repeat step 18 once. A new microcentrifuge tube can be
used for the second elution step to prevent dilution of the first eluate.
Alternatively, the microcentrifuge tube can be reused for the second elution step
to combine the eluates. See “Elution”, page 21. Note: More than 200 μl should
not be eluted into a 1.5 ml microcentrifuge tube because the DNeasy Mini spin
column will come into contact with the eluate.
Although this has yielded good results when such samples are applied to GeneChips, there is certainly
room for further development. Possible improvements could be: inclusion of dried AP1 solutes into the
solvent system and use of a solvent system (perhaps broken into stages) incorporating the following
solvents (in order of polarity): n-heptane (straight chain C7), toluene (methyl benzene), MEK (2-butanone,
methyl-ethyl-ketone), ethanol. MEK is suggested as the ketone because it is less volatile than acetone.
Heptane is preferred over hexanes because it is less volatile and anecdotally less toxic. Benzene (although
a great solvent) should be avoided for toxicity, volatility, carcinogenesis, and water-miscibility. Addition of
the solvents with some pi-activity (benzenes, ketones) are expected to act like scissors to semi- or nonpolar- substrates (such as cuticle-wax or polyphenol- laden plant cell walls).
Page 5 of 11
Matthew Lyon – Rooselab - GeneChip Protocol for DNA hybridizations derived from Borevitz’s Arabidopsis protocol
Claire Federici modified and used non-nucleon'ed version March 19-21, 2007
rewritten Mon Jul 23 10:51:06 PDT 2007 to include Nucleon resin steps.
THE LABELING PROTOCOL WITH ALL QUALITY MODIFICATIONS
some for criteria for input DNA in 100uL water:
good:
bad:
amount (ng)
>3010
<900
280/260
>1.56
<1.26
260/230
>0.69
<0.15
And typically samples with a strong and clean agarose gel appearance of the to-be-labeled DNA.
Most significant components with ANOVA analysis of historical data were '260/230' and 'agarose
appearance'. Analysis performed Mon Dec 10 12:06:23 PST 2007 by mL using 90 datapoints and some
clever data coding. Agarose gel can be deceptive if sample is complete denatured and haphazardly reannealed. Typically more input DNA is better, but excessive amounts of input DNA, like >6ug (e.g. 17 ug),
can be detrimental to the labeling process.
Additional Items Required:
Frozen 70% ethanol (-20degC)
Sodium Acetate, acidic pH, high molar concentration (3 Molar, pH 5.2)
100% Ethanol
ddH2O
Invitrogen - BioPrime® DNA Labeling System - Catalog. No.: 18094-011
Size: 30 Reactions –will yield 10 rxns at our concentrations.
Components Part No:
2.5X Random Primers Solution: [125 mM Tris-HCl (pH 6.8), Y01393
12.5 mM MgCl2,25 mM 2-mercaptoethanol,
750 µg/ml oligodeoxyribonucleotide primers (random octamers)]
10X dNTP Mixture: [1 mM biotin-14-dCTP, 1 mM dCTP, 2 mM dATP, Y01394
2 mM dGTP, 2 mM dTTP in 10 mM Tris-HCl (pH 7.5), 1 mM Na2EDTA]
Control DNA: [Salmon Sperm DNA, 10 µg/µl in RNAse-free, 55302
DNAse-free water]
Klenow Fragment (Large Fragment of DNA Polymerase I): [40 U/µl Y01396 Klenow Fragment
Buffer in 50 mM Potassium Phosphate (pH 7.0), 100 mM KCl, 1 mM DTT, 50% Glycerol]
Stop Buffer: [0.5 M Na2EDTA (pH 8.0)] 50690 Don't use this – it may contain something that
affects the GeneChip hybridization.
Distilled Water 50837
For the labeling reaction:
Turn the heat block to 100degC.
From the Invitrogen kit - put the water, dNTPs, and random primers in a plastic rack so they thaw to room
temp. Then mix and place on ice. Place the Klenow on ice.
1. Place 800 ng DNA (or more) in 100 µl of water in a microcentrifuge tube, and place on ice. Add 60 uL
of 2.5X Random Primers Solution. Close the tube, mix, clamp tube with safety cap, and hold it
down in the heat block @ 98-100C for 5 minutes –immediately cool on ice. This denatures the
DNA and allows the primers to anneal. This is more-‘watery’ than previous work with the kit but
the reaction seems to work better with more water present - - may be due to hitting the sweet spot
in terms of ionic concentration, pH, etc… for the enzyme.
Everything has to be kept at ice-temperature from this point on.
Page 6 of 11
Matthew Lyon – Rooselab - GeneChip Protocol for DNA hybridizations derived from Borevitz’s Arabidopsis protocol
Claire Federici modified and used non-nucleon'ed version March 19-21, 2007
rewritten Mon Jul 23 10:51:06 PDT 2007 to include Nucleon resin steps.
2. Perform the following additions on ice –
15µl 10X dNTP mixture
3 ul Klenow DNA polymerase.
42 uL ddH2O - This is more-‘watery’ than previous work with the kit but the reaction seems to
work better with more water present - - may be due to hitting the sweet spot in terms of ionic concentration,
pH, etc… for the enzyme.
Mix gently but thoroughly. Centrifuge 5 sec, transfer ~110 uL to each of two previously labeled small PCR
tubes. These should be pre-chilled in a cold block.
3. Incubate at 15°C overnight – Eppendorf thermocycler.
First chill Eppendorf thermocycler block to 4C by running INCUBATE command 4 degrees,
heater is ON.
Then place tubes in block, close lid and run CONSTNG program.
CTRL TUBE
Lid 80C
NOWAIT
AUTO
TUBE 0.2 ml
volume 50 (It won’t go to 75)
STEP1 T 15
3:03:03
STEP2 T 15
9:09:09
STEP3 T 70
0:15:00
STEP 4 HOLD 4C
END
The 70C step is to denature the Klenow.
We know that the product size is reduced by the reduced temperature by examining the size of probes
produced relative to a 100 b.p. ladder.
DAY TWO
Turn the heat block back on to 37C.
4. Unincorporated nucleotides can be separated from biotinylated-DNA probe by repeated ethanol
precipitation. Recombine the split sample into a new 1.5 ul microfuge tube. Add 15 ul of 3M
NaOAc (low pH ~5.2) solution and 400 uL 100 % EtOH. Mix by inverting tube repeatedly. The
volume of 220uL (aqueous) will work fine with the 400 uL EtOH and 15uL NaOAc.
Use a foam block to store tubes to avoid adding an excess of relatively hot thermal mass to the -80: Store
the ethanol, sodium acetate, sample mix in eppies upright in the -80 for at least two hours – more
time, even overnight, is better.
Re-glove, remove from the -80, put them in a microfuge and spin at max for 10 minutes.
Remove from microfuge, pour off supernatent, rinse pellet (should be white) with 400 uL of the 70%
ethanol chilled the previous day. Centrifuge again at high speed for 5 minutes.
Dry the pellet in the heat block for about 10 minutes.
Change heat block setting to 65C.
Page 7 of 11
Matthew Lyon – Rooselab - GeneChip Protocol for DNA hybridizations derived from Borevitz’s Arabidopsis protocol
Claire Federici modified and used non-nucleon'ed version March 19-21, 2007
rewritten Mon Jul 23 10:51:06 PDT 2007 to include Nucleon resin steps.
Dissolve pellet in 100 ul ddH2O. The heat of the sample from the heat-block can help, as does vortexing.
Don’t worry, biotin as a tag-label is damned-near bulletproof.
Centrifuge at max speed for 1 minute.
CAREFULLY TRANSFER THE SUPERNATANT AND NOT THE PELLET TO A CLEAN TUBE
LABELED IN THE MANNER YOU WISH TO HAVE THE GENECHIP FILES NAMED.
Imperative GeneChip - concentration standardization: Assess DNA concentration and quality by using
the NanoDrop. Dilute sample with water to give at least <500 ng/uL in 90 uL. Discard samples
that are <300 ng/uL.
Run a gel at 2% agarose to assess labeled DNA size.
Matt uses 1X TAE but I used 0.5X TBE. For a 30 ml gel 0.6 g agarose, 30 ml buffer, 8 ul of our Ethidium
bromide in gel. 85 V for 90 minutes.
Take two pictures of the gel, one for notebook, one for genomics center.
Verify that the following two criteria are met:
-
NanoDrop concentration was 400-500 or more and dropped nicely to 300-500 ng/uL w/o
any strangeness in the spectral curve.
labeled DNA forms STRONG smear 50-100-150-200… bp in size on gel.
If both above criteria are met, - transfer samples, picture of gel, FAU, etc on form to Genomics. c/o
Barbara Walter.
Be sure to fill the form out with the words “Barbara scan this chip twice” in a few places on the 1st
and 2nd page of the genechip form.
Note: Biotinylated targets are very stable and can be stored at -20°C for at least one year.
Page 8 of 11
Matthew Lyon – Rooselab - GeneChip Protocol for DNA hybridizations derived from Borevitz’s Arabidopsis protocol
Claire Federici modified and used non-nucleon'ed version March 19-21, 2007
rewritten Mon Jul 23 10:51:06 PDT 2007 to include Nucleon resin steps.
FOREIGN DNA SAMPLES IN WATER STARTING POINT
Do not let undergrads perform this procedure unless they’re
organic chemists.
Thu Sep 27 12:24:36 PDT 2007 Trials for nonpolar and semi-polar washes of DNA-Water samples
using the Qiagen kit.
REQUIRED:
- heptane
- diethyl ether
- AP1 Qiagen’s EDTA and SDS lysing buffer
- AP2 Qiagen’s acetic acid mixture - the acetic acid helps precipitate polysaccharides and detergents in
the procedure, also, DNA binding to silica (the second, white column) occurs better at low pH.
- AP3/E Qiagen’s binding buffer must be some sort of alcoholic chaotropic salt (guandinium) force-thedna-to-the-column mix. The E may denote ‘elution’ or ‘EDTA’ perhaps. Who knows/cares.
- AW Qiagen’s wash buffer – an alcoholic (quandinium?) wash
- 90-95-100% alcohol as final (white) column as a washing and drying agent
- ddH2O for elution of your sample
- RNAse A from the kit (for this procedure, pretreat sample with RNAse A in water – RNAse doesn’t seem
to need a strong buffering environment to do its job.)
To be performed in hood / with fresh gloves between and after solvents, etc… putting the vortex’er
and centrifuge in the hood will also reduce exposure to solvents - - keep both (and any heat blocks,
too) a distance from the stored solvents as motor brushes can initiate explosions:
THE PROCEDURE:
Don’t premix the initial reagents or let them sit overnight.
To the sample – standardized sample volume of 10uL. Get before and after procedure nanodrops to gauge
process losses w/ respect to ratios, too. therefore input dna amount is 10uLx Xng/uL = 10xX ng of DNA.
add 400uL AP1.
To the sample, add THE 130uL of AP2 and add the 675 AP3/E.
You will know you have added all three cause with the sample it’s clear, then the AP1 a bigger volume,
AP2 cloudiness, AP3/E makes it clear. Total volume at this point is sample + 1250uL.
Add 900 uL heptane. (or 800 uL just enough to keep a good air bubble at the top of the tube for vortexing.
Use of ART (filter) tips with the organic solvents can provide further control of moving the solvents around
as the filter ‘smoothes’ any overly-rapid pipetteing.).
Vortex. Allow to settle, discard top ~500 uL layer (the heptane). Setting the pipetteman to about 400 uL and
doing it twice will prevent taking up too much of the aqueous layer. Don’t remove any hyaloplasm or
precipitate.
Add 900 uL diethyl ether . (or 800 uL just enough to keep a good air bubble at the top of the tube for
vortexing. Use of ART (filter) tips with the organic solvents can provide further control of moving the
solvents around as the filter ‘smoothes’ any overly-rapid pipetteing.)
Vortex. Allow to settle, discard top ~1 mL layer (the ether). Setting the pipetteman to about 400 uL and
doing it twice will prevent taking up too much of the aqueous layer. Feel free to suck up any remaining
Page 9 of 11
Matthew Lyon – Rooselab - GeneChip Protocol for DNA hybridizations derived from Borevitz’s Arabidopsis protocol
Claire Federici modified and used non-nucleon'ed version March 19-21, 2007
rewritten Mon Jul 23 10:51:06 PDT 2007 to include Nucleon resin steps.
hyaloplasm or precipitate at the interface. Most of the heptane will go out with the ether. Letting heptane
hit the column tends to ruin it for binding. Some of the ether will go into the water layer. This makes the
subsequent centrifugation step a bit intoxicating/disgusting. (Ether may help column binding?) Further
investigation in this area should be pursued by qiagen r+d.
This is equivalent to the way the other foreign DNA samples have been processed but it should be noted
that some kit solutes may be lost to the extractions.
Apply resulting solution to QiaSpin colum, the second (white one).
Follow the Qiagen procedure from this point forward.
14. Pipet 650 μl of the mixture from step 13, including any precipitate that
may have formed, into the DNeasy Mini spin column placed in a 2 ml
collection tube (supplied). Centrifuge for 1 min at _6000 x g (corresponds to
_8000 rpm for most microcentrifuges), and discard the flow-through.*
Reuse the collection tube in step 15. 15. Repeat step 14 with remaining
sample. Discard flow-through* and collection tube.
DO the two AW washes.
DO the extra alcohol wash.
16. Place the DNeasy Mini spin column into a new 2 ml collection tube
(supplied), add 500 μl Buffer AW, and centrifuge for 1 min at _6000 x g
(_8000 rpm). Discard the flow-through and reuse the collection tube in step
17. Note: Ensure that ethanol is added to Buffer AW. See “Things to do before
starting”, page 24. 17. Add 500 μl Buffer AW to the DNeasy Mini spin column,
and centrifuge for 2 min at 20,000 x g (14,000 rpm) to dry the membrane. It
is important to dry the membrane of the DNeasy Mini spin column since residual
ethanol may interfere with subsequent reactions. This centrifugation step ensures
that no residual ethanol will be carried over during elution. Discard flow-through
and collection tube. After washing with Buffer AW, the DNeasy Mini spin column
membrane is usually only slightly colored. In the rare case that the membrane
remains significantly colored after washing with Buffer AW, refer to “Darkly
colored membrane or green/yellow eluate after washing with Buffer AW” in the
Troubleshooting Guide on page 45. Note: Following the centrifugation, remove
the DNeasy Mini spin column from the collection tube carefully so the column
does not come into contact with the flowthrough, as this will result in carryover of
ethanol.
Elute into NO MORE THAN 100 uL of ddH2O (re-elute back through to get []’s up – this works by prewetting the column with the water, whereas the second elution carries the softened/release DNA out more
efficiently) or 500 uL of ddH2O, followed by heat volatilization of the extra ddH2O only if your endpoint
can handle ssDNA (note this makes it impossible to quantify or characterize your end product, so only do
this if you’re really desperate to get as much DNA off the silica column as possible.)
18. Transfer the DNeasy Mini spin column to a 1.5 ml or 2 ml
microcentrifuge tube (not supplied), and pipet 100 μl Buffer AE directly
onto the DNeasy membrane. Incubate for 5 min at room temperature (15–
25°C), and then centrifuge for 1 min at _6000 x g (_8000 rpm) to elute.
Elution with 50 μl (instead of 100 μl) increases the final DNA concentration in the
eluate significantly, but also reduces overall DNA yield. If larger amounts of DNA
(>20 μg) are loaded, eluting with 200 μl (instead of 100 μl) increases yield. See
“Elution”, page 21. 19. Repeat step 18 once. A new microcentrifuge tube can be
Page 10 of 11
Matthew Lyon – Rooselab - GeneChip Protocol for DNA hybridizations derived from Borevitz’s Arabidopsis protocol
Claire Federici modified and used non-nucleon'ed version March 19-21, 2007
rewritten Mon Jul 23 10:51:06 PDT 2007 to include Nucleon resin steps.
used for the second elution step to prevent dilution of the first eluate.
Alternatively, the microcentrifuge tube can be reused for the second elution step
to combine the eluates. See “Elution”, page 21. Note: More than 200 μl should
not be eluted into a 1.5 ml microcentrifuge tube because the DNeasy Mini spin
column will come into contact with the eluate.
Page 11 of 11