Plant Genomic DNA Isolation Protocols

Fix plant tissue with absolute alcohol

Put plant leaves in fixing solution absolute alcohol for 30 min to denature enzymes at room temperature (RT), allow alcohol to evaporate. Homogenize dried leaves with a mortar and pestle

DNA extraction protocol

A. Breaking the Plant Cell Wall and Cell Lysis

(The purpose of this step is to break down the cell wall of the plant. It is thus important to get the plant tissue very well ground up, but this procedure needs to be done quickly to prevent your DNA from degrading during the procedure.)

1. Cut frozen plant tissue into pieces as fine as possible with a razor blade. In mortar, add 4-10 leaves of cut up plant tissue and 5 ml of CTAB reagent (100mM Tris pH= 8.0, 1.4M NaCl, 25 mM EDTA, 2% CTAB, 0.2% beta-mercaptoethanol). Grind tissue using pestle to yield a fine slurry.

2. Add 15 ml more CTAB buffer to the mortar and continue to grind until of uniform texture. Pour material into a 50ml centrifuge tube. Use a rubber policeman to transfer as much material as possible. Clean the mortar and pestule and repeat with second plant. Transfer the next slurry into a different 50ml microfuge tube. Continue to repeat procedure until all samples are ground up. (Make sure you label all 50ml tubes.)

3. Place the tubes in a 50 C water bath for approximately 1 hour. (The Active ingredient in this process is the detergent, CTAB. EDTA has been added to deter Dnase activity.)

4. Spin Tube down at 1,300 X G for 2 minutes to get rid of sand and debree. Transfer the supernatent to a new 50 ml tube and discard sand and debree into trash.

B. DNA Extraction: chloroform/isoamyl alcohol (or octanol) (24:1) Two important chemicals are used in this step, Chloroform and alcohol. Each chemical is used to help separate proteins and polysaccharides from nucleic acids in the cell. Chloroform is more dense than water solutions, thus after spinning this solution, the chloroform and water will separate into two distinct phases. The lower phase will be Chloroform/alcohol. This is the phase that proteins and polysaccharides find most chemically attractive. The upper aqueous phase will contain your DNA. With plant tissue there are often more polysaccharides than in animal tissue. If the polysaccharides are not removed, many enzymatic procedures such as PCR or restriction enzyme digests will not work appropriately. These steps should be performed in the fume hood.

5. Add 25 ml of chloroform/octanol.

6. Mix by inverting tube several times. Make sure that the lid is on tight.

7. Centrifuge at 10,000xG (or max on other centrifuge) for 2-3 minutes.

8. Check the color of the aqueous layer.

If it is clear= Without disturbing the lower green level (chloroform) or the middle whitish layer (various organics and parts of leftover plant tissue), transfer the upper clear colored liquid into a fresh 50 ml tube and continue onto the steps below. Follow your instructors guide in disposing of chloroform properly.

If it is cloudy= Without disturbing the lower green level (chloroform) or the middle whitish layer (various organics and parts of leftover plant tissue), transfer the upper cloudy colored liquid into a fresh 50 ml tube, add more CTAB to a volume of 20 ml and then redo steps 5, 6,7 and 8. Follow your instructors guide in disposing of chloroform properly

C. DNA Precipitation

9. Add an equal volume (approx 25ml) of ice cold isopropanol to your sample. Mix the tube gently by inversion until DNA precipitates (5-10 min.) Place on ice for 10 minutes. (if you have a lot = pull out the pellet of DNA and place it in a 2 ml tube and go to step 11a, if you don't have a visible pellet go to step 10).

10. Centrifuge the tube at 10,000 X G for six minutes (or two times 3 minutes if you need to work around students who are still trying to get clear aqueous layers) to pellet the DNA. Now go to step 11b.

11a. Dissolve pellet in approximately 1000ul of TE. Mix by inversion and place in the 50 degree incubator until completely dissolved or you have tried to dissolve it for at least 10-15 minutes. Transfer 500 ul of solution to a new 2 ml microfuge tube and proceed to step 12.

11b. Dump supernatant. Dissolve pellet in approximately 500ul of TE. Mix by inversion and place in the 50 degree incubutar until completely dissolved or you have tried to dissolve the pellet for at least 10 min. Transfer 500 ul of solution to a 2 ml microfuge tube and proceed to step 12.

12. Add 1/10 volume (about 50ul) of 7.5M ammonium acetate or 3M sodium acetate (pH= 5.2) and 2.1 volumes of ethanol (about 1100ul). Mix

13. Centrifuge for 10 minutes in the microfuge at maximum speed

14. Dump the supernatant, then wash the pellet with about 400 ul of 70% Ethanol. This will get rid of any excess salt.

15. Centrifuge for 5 minutes at maximum speed.

15. Discard the supernatant and centrifuge for 30 seconds. Remove all supernatant with a pippette tip and then allow the pellet to air dry until all traces of ethanol are removed.

16. Resuspend the DNA in about 700 ul of TE buffer.

17. Rehydrate DNA by incubating sample overnight at room temperature. The sample should be placed in the refrigerator the next day.

Another protocol

1. Preheat the extraction buffer containing 100 mM Tris–HCl (pH 8), 1.4 M NaCl, 20 mM EDTA (pH 8), 2% (w/v) CTAB in water bath at 60°C for about 15 minutes.
2. Submerge 1 g of plant tissue in 5 ml of absolute alcohol for 5 minutes and allow alcohol to evaporate.
3. Grind the tissue in presence of 1% PVP (Polyvinylpyrrolidone) and pre-warmed extraction buffer by using a pre-chilled mortar and pestle (−40°C/−80°C) at room temperature.
4. Transfer the ground material into 2 ml centrifuge tubes and incubate in water bath at 60°C for 1 hour.
5. Centrifuge the tubes at 10,000 rpm for 10 minutes at 4°C and collect the supernatant in 1.5 ml centrifuge tube using wide bored tip.
6. To the supernatant add equal volume of chloroform:isoamyl alcohol (24:1) and mix by inversion for 15 minutes.
7. Centrifuge the tubes at 10,000 rpm for 10 minutes at 4°C and collect the supernatant in 1.5 ml centrifuge tube.
8. Again add equal volume of chloroform: isoamyl alcohol (24:1) to the supernatant and mix by inversion for 15 minutes.
9. Centrifuge the tubes at 10,000 rpm for 10 minutes at 4°C and collect the supernatant.

10. To the supernatant add twice the volume of chilled isopropanol to precipitate the DNA and incubate it at −20°C for 30 minutes.
11. Centrifuge the tubes at 10,000 rpm for 10 minutes at 4°C and collect the pellet.
12. Wash the pellet 2 –3 times with 70% ethanol and air dry the pellet in room temperature.
13. Add 50–100μl of TE buffer to dissolve the DNA.
14. Store at −20°C for further use

Reagents

100 mM Tris-HCl (pH 8)

1.4 M Sodium Chloride (NaCl)

20 mM-Ethylenediaminetetraacetic acid (EDTA) (pH 8)
2% (w/v) Cetyl trimethylammonium bromide (CTAB)
Chloroform-isoamyl alcohol (24:1)
Isopropanol

70% ethanol
TE buffer (pH 8): 10 mM Tris–HCl, 1 mM EDTA

Silica  powder (Celite 545 silica) method

Materials

Celite 545 silica powder (Celite 545-AW reagent grade) Sigma 20199-U
Buffers

DNA BINDING BUFFER	6M Guanidine thiocyanate MW = 118.2 g/mol 70.92 g / 100mL (6M)	!!! it takes several hours until dissolved (leave it approx. 4-5 hours)
WASH BUFFER	1mL of 5M NaCl + 99mL of 95% EtOH	!!! PREPARE FRESH, because the salt precipitates during storage
LYSIS BUFFER (standard)	0.5% SDS (w/v) in 10x TE 0.5g SDS /100mL
5M NaCl stock solution	MW=58.44g/mol 29.22g / 100mL	Do not use if percipitate forms. Either heat to get fully back into solution or 5M NaCl stock solution discard and make fresh
3M Sodium acetate (pH = 5.2)	MW=82.03g/mol 24.61g / 100mL	Adjust pH value with glacial acetic acid
DNA ELUTION BUFFER	Tris-EDTA (TE) buffer, 10mM Tris-HCl, pH=8.0, 1 mM EDTA
10X TE	Composition: 100mM Tris-HCl, pH=8.0 10 mM EDTA

PREPARATION OF SILICA POWDER-DNA BINDING SOLUTION

Fill silica powder (Celite 545 silica) into 50 mL Falcon-tube (to about 2.5mL = approx. 800mg)
Add 30 mL dH2O
Shake vigorously (vortex and invert)
Let slurry settle for approx. 15 min
Remove (pipette off) the liquid
Repeat 2 times (a total of 3 washes)
After last washing step: resuspend the silica powder in about the same amout of water(up to about 5 mL)

STORE the silica solution at RT until further use (silica : H 2 O = 1 : 1)
Before use:
suspend stored silica solution (silica : H 2 O = 1 : 1) by vortexing
Transfer ~50 μL of silica solution to 2mL tubes (prepare 1 tube per sample)
!! try to keep the silica suspended during pipetting to ensure an equal distribution
Add 1mL H2O (a final wash step)
Mix by vortexing
Centrifuge: full speed (13,200) for 10-20 sec
Pipette off liquid
Add 700 μL DNA binding buffer (6M Guanidine thiocyanate)
Suspend the silica powder in DNA binding buffer
The silica binding solution is now ready for further use in the protocol (see Methods)

PREPARATIONS
 Prepare 2 mL tubes (1 per sample): add 3 metal beads (tungsten carbide beads) per tube
 Harvest leaf material (starting amount of material: about 100 mg fresh weight)

GRINDING
Use appropriate / available grinding protocol (Mortar & pestle, Qiagen TissueLyser)
e.g. grinding using Qiagen TissueLyserII:
 Freeze 2mL tubes containing leaf material and 3 metal beads in liquid nitrogen
 Grind in TissueLyser by shaking (10 sec at 1/30 speed)
 Re-freeze in liquid nitrogen (>30 sec)
 Grind again in TissueLyser by shaking (10 sec at 1/30 speed)
 Re-freeze in liquid nitrogen (>30 sec)
 Keep in liquid nitrogen until lysis buffer is added or store at -80°C for later use

LYSIS
 Add 800 μL Lysis buffer
 Add 4 μL RNaseA (100 mg/ml)
 Vortex (~2 min until the powder is dissolved in the buffer)
 Incubate: 10min at room temperature
 Add 200 μL 3M Sodium Acetate (pH = 5.2)
 Mix by inversion of tubes
 Incubate on ice for 5 min
 Centrifuge 13,200 rpm / 5 min / RT (pellet the leaf material)

DNA BINDING
 prepare 700 μL silica binding solution (see above)
 transfer 800 μL of the supernatant to the tubes containing silica binding solution)
!! Do not transfer leaf material!
 Completely resuspend the silica powder by vortexing and inversion of tubes (ca 20 sec)
 incubate 15 min at RT (on a shaker at 400 rpm and/or invert tubes from time to time)
 Centrifuge 13,200 rpm / 3 min / RT (pellet the silica)
 Remove the supernatant (with pipette)
WASHING (2 times)
 Add 500 mL wash buffer (prepared fresh)
 Completely resuspend the silica powder by vortexing and inversion of tubes (approx. 20 sec)
 Centrifuge 13,200 rpm / 3 min / RT (pellet the silica)
 Repeat the washing step (optional: a third washing step)
 Remove the supernatant with pipette (as complete as possible)
 optional: short spin and remove residual liquid with pipette
 After last washing step: dry the silica in the hood up to 1 hour at RT (make sure there is no wash buffer left)

RESUSPENSION
 Add 200μL TE buffer or 10mM Tris buffer
 Completely resuspend the silica powder by vortexing and inversion of tubes (approx. 20 sec)
 Incubate: 20 min / RT / with gentle agitation (on a shaker at 400 rpm and/or invert tubes from time to time)
 Centrifuge (for tubes): 13,200 rpm / 5 min / RT (pellet the silica)
 transfer 180 μL supernatant to new tube (avoid transferring silica powder!)
 optional: if there is still silica powder in the preps – repeat the centrifugation
 check for concentration and integrity of DNA
 store the genomic DNA at -20°C for long-term storage or 4°C for short-term storage

Modified Qiagen Protocol

- 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-the-dna-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.

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.

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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 ready-to-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 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.

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. 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 pre-wetting 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 non-polar- substrates (such as cuticle-wax or polyphenol- laden plant cell walls).