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.
this procedure may assist people working with other sensitive procedures in 'difficult' (read: interesting
? ; non-Arabidopsis / non-young-barley) species. the goal is to obtain DNA in a relatively highthroughput procedure that is of reasonably high-concentration and of a quality level that leaves one notquestioning the quality of their initial DNA sample. in the old days, cesium chloride ultracentrifugation might be used to a similar endpoint but would be limited to 5-6 samples a night.
GeneChip work status: Mon Jul 23 14:22:16 PDT 2007. We know that the biotin is being incorporated by examining a gel pre-stained with the stravidin-flurophore dye. A region of 'clearing' is noted before the region associated with DNA that fluoresces. Upon subsequent addition of EtBr staining the freshly produced DNA is noted in this region, as well. Further, the ladder is without fluorescence from the SA dye, providing a negative control for the gel visualization step.
The age of chip or sample utilized doesn't seem to affect results because we've compared old versus new samples and re-done old samples. The scanner also doesn't seem to affect things because we've compared the calls from the UC Irvine Genomics facility versus the UCR Genomics Facility scanner.
We are investigating the removal of polysaccharides and non-inclusion of any acetate ions in the input steps of the amplification reaction as adversely affecting the labeling procedure. Anecdotal evidence of adverse effects of polysaccharide contamination to Klenow were shared by veteran molecular biologist
Timothy Close. The internet provided the anecdote that Klenow is negatively impacted by large quantities of acetate ion. An acetate precipitation step was utilized to 'help' purify the DNA before the labeling procedure in some earlier labeling reactions. Some of these reactions failed. Recent attempts to force this
failure mode also support this hypothesis (process QA). In-sample contaminants are also being investigated: because the samples that fail more on the GeneChip platform were extracted using the CTAB procedure at a site other than UCR the presence of polysaccharides as a contaminant is suspected in these samples. It is very difficult to remove polysaccharides, or carbohydrates, from DNA samples in water. For this reason we are investigating a procedure that removes non-polar tissue components and polysaccharides early in the extraction procedure to avoid complicating the downstream labeling and
GeneChip hybridization steps. For this we are trying the Nucleon Phytopure borated poly-resin and chloroform as additives to the initial steps in the Qiagen procedure.
For DNA extraction: Prewarm dry bath to 65C.
Acquire the following:
2 ml microfuge tubes. ice bucket. dry ice for ground leaves. liquid N2.
1 mortar, pestle and spatula for each sample. container to store extra ground leaves.
Qiagen kit.
Bioprime kit
Phytopure kit SL8510 Nucleon PhytoPure kit for 50 extractions of 0.1g of plant tissue
Chloroform (be careful with this stuff - - it is both intoxicating and turns into the poison gas 'phosgene' when exposed to light, heat, or mixed biological enzymes.)
Page 1 of 13

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 Phytopure resin and/or Qiagen final (white) silica column are primarily dependent on the composition
(especially the 'ionic strength') of solutions for binding and/or elution. This procedure matches the volumes of the appropriate solution from both kits so that the resin or column steps have an applied-solution- composition consisting of one-half the typical components at this step. This final procedure was decided upon after a few different arrangements of the various steps from both procedures. With this 'premix version', the chloroform into an emulsion containing some alcohol, which reduces its breakdown and volatilization, making it safer and easier to use.
Wash the collected leaves 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.
6. This shouldn't have to be stated: but: Work in a hood with gloves and face shield or goggles throughout the chloroform containing portions of this protocol. To avoid contaminating lab surfaces or the handle of the -80 with the chloroform DNA extraction mixture please re-glove after leaving the hood or lab to reaccess the -80 freezer. The number of trips to the -80 can be reduced by recovering the samples in bulk from the -80, keeping them local and frozen using dry ice.
Into a master mix, mix in a Fisher tube multiplied by two-more than the number of samples:
- 500 uL chloroform
- 400 uL buffer AP1 (Qiagen – make sure ethanol has been added)
- 100 uL Amersham Nucleon Phytopure resin (must vortex before pippeting).
- 130 uL AP2 (Qiagen)
- 200 uL Amersham Reageant 2
- 600 uL Amersham Reageant 1
- mix by a quick vortex and again before using 1 mL of it per sample. should form a mocha looking colored emulsion with small brown blobs (the resin) that gradually glob up and try to come out of the emulsion.
7. Grind the sample of young leaves that under liquid N2 until very fine (usually three applications of nitrogen). DO NOT ATTEMPT TO USE DRY ICE as the freezing agent as suggested in the Amersham manual - - it just ends up turning your sample into a semi-explosive (carbonated) chloroform emulsion and is both messy and dangerous. After the third grind add 1 mL of the above prepared emulsion and another quick shot of liquid nitrogen to grind a bit of the extraction emulsion in with the tissue. Add 4 ul Rnase A stock solution (100 mg/ml) into a 1.7 or 2 mL eppie with 1 mL of the emulsion and 100 mg of the powdered leaf tissue to the emulsion in the tube. If the initial tissue disruption does not produce a homogenate of small plant tissue pieces, the sample can sometimes be 'saved' by a quick vortex, freezing, thawing and re-vortexing of the sample. If this is attempted care must be taken to avoid having the chloroform-containing sample explode upon freezing or thawing.
8. Quickly vortex the sample, open the tube , still in the hood: incubate the mixture for 10 min at 65°C then, seal the tube, and quickly vortex the sample. The reason to be in the hood for this part of the procedure is that the chloroform will boil a bit at this stage in the procedure because of its high vapor pressure at this temperature.
10. Centrifuge the lysate for 10 min at max: 20,000 x g (~14,000 rpm).
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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.
Pour off the supernatant into a fresh, labeled eppie.
13. Gently, without disturbing the plant debris and resin strata, add 675 µl Buffer AP3/E on top of the pellet and carefully pour this as well into the new eppie. Mix this new eppie by inversion. Try not to get any brown resin or plant scraps into the new eppie. Properly discard this pellet, resin, chloroform, and plant debris containing tube. (If desired the chloroform-y pellet it can be examined under the microscope before or after drying.)
Because the majority of the chloroform has been contained in the plant debris and heavy layers, the remaining portions of the procedure may be performed at the bench, out-of- the hood. For the safety of yourself and others: change your gloves at this point.
14. Pipet 650 µl of the mixture from step 13, 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. *Flow-through fractions contain Buffer AP3/E, and are therefore not compatible with bleach.
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 of Dneasy protocol.
17.Add 500 µl Buffer AW to the DNeasy Mini spin column, and centrifuge for 2 min at at >6000 x g
(>8000 rpm). After washing with Buffer AW, the DNeasy Mini spin column membrane is usually less colored than with the Qiagen-only procedure. Following the centrifugation, remove the
DNeasy Mini spin column from the collection tube carefully so the column does not come into contact with the flow-through, as this will result in carryover of ethanol. We know that this step actually washes the DNA and helps produce a clean band based on the examination at the end of the procedure of Qiagen-purified DNA (appears the cleanest of all methods using agarose gel electrophoresis.)
17 extra. Add 500 µl 96-100% EtOH to the DNeasy Mini spin column, and centrifuge for 2 min 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.
18. Transfer the Dneasy Mini spin column to a 1.5 ml microfuge tube, and pipet 500 ul water directly onto the membrane, Incubate 5 minutes at room temperature then centrifuge 1 minute at >6000 x g
Page 3 of 13

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.
(>8000 rpm). Remove column from tube and pipette the eluent back onto the column. Again incubate 5 minutes at room temperature then centrifuge 1 minute at >6000 x g (>8000 rpm).
19. DNA Polymerase I, Klenow fragement is a not an especially robust laboratory enzyme with respect to contaminant tolerance or thermal stability. For this reason, avoid concentrating the DNA at this step with an ethanol and acetate precipitation step (would add acetate). The sample can be concentrated using heat and/or vacuum until you have about 40-50 uL of concentrated DNA solution. If heat is applied, expect to see light smear centered around 500-1000 bp in size. If the
DNA got excessively sheared (perhaps mechanically) a strong smear centered around 500-1000 bp in size. If you want to see your DNA intact use 100 uL of the ddH2O for the elution.
Quantify the DNA using the NanoDrop. Save 5ul for a gel later.
Supplying the labeling reaction with an excess of DNA doesn't affect the final product size ranges for the amplification(labeling) step. This was investigated in a quantitative manner by acetate ionhindering the labeling reaction across a range of greatly increased template concentrations - - all labeling reactions produced equivalent in product size ranges, although those with extremely increased quantities of input DNA produced greater quantities of labeled target.
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
- 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.
Page 4 of 13

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.
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.
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 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 demyelization of nerve tissue), 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.
Page 5 of 13

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.
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 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 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
Page 6 of 13

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.
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.
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.
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.
Page 7 of 13

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.
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 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 8 of 13

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.
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.
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 ~ 75 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 CONST15 program.
CTRL TUBE
Lid 80C
NOWAIT
AUTO
TUBE 0.2 ml
Page 9 of 13

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. volume 50 (It won’t go to 75)
STEP1 T 15
STEP2 T 15
STEP3 T 70
3:03:03
9:09:09
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 solution and 400 uL 100 % EtOH. Mix by inverting tube repeatedly.
Place at -20 for 1 hour then -80 for 30 minutes. (use a foam block to store tubes to avoid adding an excess of relatively hot thermal mass to the -80.)
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 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.
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 300 ng/ul in 90 uL of water.
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 ng/uL w/o any strangeness in the spectral curve.
labeled DNA forms STRONG smear 50-100-150-200… bp in size on gel.
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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.
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 11 of 13

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.
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:
THE PROCEDURE:
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 1 mL heptane.
Vortex. Allow to settle, discard top 1 mL layer (the heptane).
Add 1 mL diethyl ether
Vortex. Allow to settle, discard top 1 mL layer (the ether). 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.*
Page 12 of 13

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.
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 starti ng”, 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 (n ot 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 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.
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