Simple DNA extraction from plants

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Anthony Liekens

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Sep 1, 2009, 7:42:40 AM9/1/09
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In building my first DIY biolab, I'm looking into doing RAPD
experiments on helleborus plants. I'll be adopting a thermal cycler
from work, and am now making a shopping list of equipment to buy or
build (pipettes, scale, vortex, centrifuge, electrophoresis chamber)

I'm quite troubled finding tested and proven but easy and inexpensive
protocols for extracting DNA from plant material. There's just too
many protocols to choose from. In the literature, some one-step
protocols simply add plant material to a lysis buffer and that's that,
where other more elaborate techniques require more than a day's work.

Has anyone on the mailing list been extracting DNA from plants to do
PCR amplification? Which technique works best in a lab-in-the-garage
environment? I'm looking for a minimal approach that doesn't require
expensive materials and equipment (e.g., I have no access to liquid
nitrogen). I am willing to try a few protocols and reporting on their
quality.

Thank you,

Anthony,-

Ben Gadoua

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Sep 1, 2009, 2:22:59 PM9/1/09
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Anthony;

   I'm not sure what reagents you can get, however the protocol below is rather complex, needs a highspeed rotor, and is probably not the healthiest for you but here goes:


Prepare plant tissue

1. Harvest 10 to 50 g fresh plant tissue.

Plants may be placed in the dark for 1 to 2 days prior to harvest to reduce the starch content

in the tissues.

Younger plants are the preferred source of tissue because they have a lower polysaccharide

content.

2. Rinse tissue with deionized water to remove adhering debris and blot dry.

3. Freeze tissue with liquid nitrogen and grind to a fine powder in a mortar and pestle.

Keep the tissue frozen throughout this procedure by occasionally adding liquid nitrogen.

4. Transfer frozen powder to a 250-ml centrifuge bottle and immediately add 5 to 10 ml

extraction buffer per gram fresh plant tissue. Stir gently to disperse tissue.

5. Add 10% N-lauroylsarcosine to a final concentration of 1%. Incubate 1 to 2 hr at

55°C.

It is important to add N-lauroylsarcosine after the tissue is resuspended in extraction buffer.

If N-lauroylsarcosine is included in extraction buffer, premature lysis of the plant cells will

interfere with tissue dispersal and lead to unwanted shearing of DNA.

The lysate should be clear, green, and slightly viscous. From this point on solutions should

be handled gently to reduce shearing of the DNA—use a wide-bore pipet and do not vortex

or mix vigorously.

6. Centrifuge lysate 10 min at 5500 × g (6000 rpm in a Beckman JA-14 rotor), 4°C, to

pellet debris. Save the supernatant and centrifuge again if necessary to remove

undigested debris.

Precipite the DNA

7. Add 0.6 vol isopropanol to the supernatant and gently mix. A nucleic acid precipitate

should be visible; if not, incubate 30 min at 20°C.

8. Centrifuge 15 min at 7500 × g (8000 rpm in a Beckman JA-14 rotor), 4°C. Discard

supernatant.

Do not let the nucleic acid pellet dry or it will become extremely difficult to dissolve.

Carry out CsCl centrifugation

9. Resuspend pellet in 9 ml TE buffer. If necessary, incubate at 55°C to aid resuspension.

Add 9.7 g of solid CsCl and mix gently until dissolved.

To minimize depurination, limit 55°C incubation to 2 hr.

10. Incubate 30 min on ice. Centrifuge 10 min at 7500 × g (8000 rpm in a JA-20 rotor),

4°C, and save supernatant.

This clearing spin removes some of the insoluble debris remaining in the lysate. In addition,

a small separate phase may form on the top of the solution after centrifugation; this is due

to residual Sarkosyl in the lysate. The Sarkosyl phase can be removed by filtering the

supernatant through two layers of cheesecloth. Collect the supernatant but discard the

Sarkosyl phase.

11. Add 0.5 ml of 10 mg/ml ethidium bromide and incubate 30 min on ice.

CAUTION: Ethidium bromide is a mutagen. Be careful and wear gloves.

12. Centrifuge 10 min at 7500 × g, 4°C.

A large RNA pellet should form. At this point much of the unwanted constituents in the

lysate—RNA, protein, and carbohydrates—have been removed.

13. Transfer the supernatant to two 5-ml quick-seal ultracentrifuge tubes and seal tubes.

Make sure tubes are full, balanced, and well-sealed.

14. Centrifuge 4 hr at 525,000 × g (80,000 rpm in a Beckman VTi80 rotor), 20°C, or

overnight at 300,000 × g (60,000 rpm in VTi80 rotor), 20°C.

Collect and purify DNA

15. Gently remove the tube. Punch a hole in the top (to provide an air inlet) with a

large-bore (15-G) collecting needle. Recover the DNA band by inserting needle,

attached to a 1-ml syringe, through tube wall directly below the band (see Fig. 1.7.1).

This operation is identical to that used during plasmid purification, except that only one

band should be visible.

CAUTION: If UV illumination is used to visualize the DNA, wear UV protective glasses

or a face shield. Minimize exposure of gradient to visible light to reducing nicking of DNA

caused by ethidium bromide.

16. Remove the ethidium bromide by repeatedly extracting the collected DNA with

CsCl-saturated isopropanol.

17. Add 2 vol water and 6 vol ethanol to the DNA solution and mix. Incubate 1 hr at

20°C.

DNA may precipitate immediately as a single white mass; it can be collected using a Pasteur

pipet with a hook introduced at the tip or by brief centrifugation.

18. Centrifuge 10 min at 7500 × g, 4°C.

19. Resuspend pellet in TE buffer and reprecipitate DNA by adding 110 vol of 3 M sodium

acetate and 2 vol ethanol. Incubate at 20°C if precipitate is not visible and collect

DNA by centrifugation.

20. Briefly air dry the final pellet and resuspend in 0.5 to 2 ml TE buffer.

A DNA concentration of 100 ng/_l is generally convenient for most purposes.

 

Extraction buffer

100 mM TrisCl, pH 8.0

100 mM EDTA, pH 8.0

250 mM NaCl

100 ìg/ml proteinase K (add fresh before use)

Store indefinitely at room temperature without proteinase K

High-salt TE buffer

10 mM TrisCl, pH 8.0

0.1 mM EDTA, pH 8.0

1 M NaCl

Store at room temperature (stable for several years)


I'm sure I can come up witha d ifferent protocol if you don't have access to highspeed rotors, or if the cscl2 gradient seems like a bit of a pain, but that's the one that I have handy right now.


Ben


Nathan McCorkle

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Sep 1, 2009, 3:07:20 PM9/1/09
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On Tue, Sep 1, 2009 at 7:42 AM, Anthony Liekens <anthony...@gmail.com> wrote:

In building my first DIY biolab, I'm looking into doing RAPD
experiments on helleborus plants. I'll be adopting a thermal cycler
from work, and am now making a shopping list of equipment to buy or
build (pipettes, scale, vortex, centrifuge, electrophoresis chamber)


You should have, in addition to a thermal cycler, a centrifuge, autoclavable (polypropylene) tissue grinders (small mortar and pestle), I recommend getting p10, p200, and p1000 pipettes, a hot plate, a thermometer, and maybe a vortexer, as well as pipet tips.
 
I'm quite troubled finding tested and proven but easy and inexpensive
protocols for extracting DNA from plant material. There's just too
many protocols to choose from. In the literature, some one-step
protocols simply add plant material to a lysis buffer and that's that,
where other more elaborate techniques require more than a day's work.

 
Has anyone on the mailing list been extracting DNA from plants to do
PCR amplification? Which technique works best in a lab-in-the-garage
environment? I'm looking for a minimal approach that doesn't require
expensive materials and equipment (e.g., I have no access to liquid
nitrogen). I am willing to try a few protocols and reporting on their
quality.

Yes I did it a few times this spring, it is quite simple, basically you grind plant tissue (sterile grinders), add a bit of lysis buffer (as pure liquid soap (SDS based) as you can find, maybe adjust the pH, and add EDTA and some wide-spectrum protease (meat tenderizer maybe, bromelain) if you can), grind some more, add a bit of non-soapy buffer and transfer to a micro test-tube, incubate at hot temp for 5-60 minutes, centrifuge on high speed to pellet cell wall/plant structure materials, decant/pipette and save the supernatant (containing DNA and some proteins), then add ethanol to get the DNA out of solution and centrifuge it to the bottom, then discard the solution, dry remaining ethanol by letting the tube sit out (or place in a heater on low temp) then resuspend in pH adjusted buffer, maybe with some EDTA.

There are a lot of variations you can do, but a simple grinding, adding some soapy water, heating a bit, centrifuging to collect liquid, adding ethanol, centrifuging to dispose liquid, drying ethanol, and resuspending in even pure water.... WILL WORK... just maybe the yield will be low.

I have a lab report for my plant molecular biology class that I have at home somewhere, I can try to get it to you in the next day or so.
 

Thank you,

Anthony,-

Welcome,
-Nathan
 





--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

Nathan McCorkle

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Sep 1, 2009, 3:11:48 PM9/1/09
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On Tue, Sep 1, 2009 at 2:22 PM, Ben Gadoua <ben.g...@gmail.com> wrote:
Anthony;

   I'm not sure what reagents you can get, however the protocol below is rather complex, needs a highspeed rotor, and is probably not the healthiest for you but here goes:

I'm sure I can come up witha d ifferent protocol if you don't have access to highspeed rotors, or if the cscl2 gradient seems like a bit of a pain, but that's the one that I have handy right now.


CsCl2 doesn't seem neccesary in this case, as long as you have plenty of plant material, you can get nice DNA with soap, water, and ethanol.


Ben




Ben Gadoua

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Sep 1, 2009, 3:25:45 PM9/1/09
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Nathan;


   The soap method isn't very purified, there's much plant material, RNA, and other contaminants that make it extremely difficult to do competitive PCR. For proper competitive PCR you need to be have pure DNA with normalized concentrations eg. plant A has 300ng/ul of dna but and a A260 reading is 10, and the A260/A280 ratio is .5 and the A260/A230 ratio is .7, the dna is not very pure, and the A260 reading(and therefore concentration) is most likely artificially inflated, plant B has a concentration of 150ng/ul with an A260 reading of 5, the A260/A280 reading 2 and the A260/A230 ratio is 2, you most likely have very pure DNA of the concentration given. The CsCl2 gradient is a method for seperating the DNA from the plant material that is found in impure extractions. If you try to run competetive PCR with inpure DNA you will not be able to quantify it at all, the bands on your gel won't have a uniform response to EtBr or to SYBRGreen. Even if you do them relative to a reference gene (B2M, GAPDH, etc) the results will not be consistent, there are other methods for DNA purification after extraction, this is an older one, and I'm sure I can find a better one somewhere, this is just the one that I had handy.

Ben

Nathan McCorkle

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Sep 1, 2009, 7:39:32 PM9/1/09
to diy...@googlegroups.com
On Tue, Sep 1, 2009 at 3:25 PM, Ben Gadoua <ben.g...@gmail.com> wrote:
Nathan;


   The soap method isn't very purified, there's much plant material, RNA, and other contaminants that make it extremely difficult to do competitive PCR. For proper competitive PCR you need to be have pure DNA with normalized concentrations eg. plant A has 300ng/ul of dna but and a A260 reading is 10, and the A260/A280 ratio is .5 and the A260/A230 ratio is .7, the dna is not very pure, and the A260 reading(and therefore concentration) is most likely artificially inflated, plant B has a concentration of 150ng/ul with an A260 reading of 5, the A260/A280 reading 2 and the A260/A230 ratio is 2, you most likely have very pure DNA of the concentration given. The CsCl2 gradient is a method for seperating the DNA from the plant material that is found in impure extractions. If you try to run competetive PCR with inpure DNA you will not be able to quantify it at all, the bands on your gel won't have a uniform response to EtBr or to SYBRGreen. Even if you do them relative to a reference gene (B2M, GAPDH, etc) the results will not be consistent, there are other methods for DNA purification after extraction, this is an older one, and I'm sure I can find a better one somewhere, this is just the one that I had handy.

Where did he mention wanting to do competitive PCR?? All I see him mentioning is RAPD PCR, which doesn't require exact amounts of DNA at all.
Ben



Cory Tobin

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Sep 1, 2009, 7:49:28 PM9/1/09
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On Tue, Sep 1, 2009 at 4:42 AM, Anthony
Liekens<anthony...@gmail.com> wrote:
> Has anyone on the mailing list been extracting DNA from plants to do
> PCR amplification? Which technique works best in a lab-in-the-garage
> environment? I'm looking for a minimal approach that doesn't require
> expensive materials and equipment (e.g., I have no access to liquid
> nitrogen). I am willing to try a few protocols and reporting on their
> quality.

The basic protocol I use is 1) freeze 2) crush 3) filter out large
debris 4) phenol-chloroform extraction + ethanol precipitation

1) For the first step I use liquid nitrogen, but you might be able to
get away with just putting the samples in an ordinary freezer for a
while, along with your mortar and pestle. Another alternative I
thought of (but have never tried) is to freeze using one of those "air
in a can" dusters. PopSci has a video tutorial on freezing a can of
soda with one of those things. http://www.popsci.com/node/30587 I'm
not sure how cold it gets or how safe it is, but it may work.

2) For crushing I use a 1.7mL eppendorf tube in place of a mortar, and
a small pestle. Both are chilled to the same temp as the sample.

3) I'm not sure if the filtering step is necessary. Lots of protocols
I have seen neglect this step, but I do it anyways (it makes the
phenol chlorform extraction easier). To do this I first add 1mL
extraction buffer to the ground tissue and mix up and down with a
pipette. Buffer = 100mM Tris, 50mM EDTA, pH 8.0. Then I put some
cheesecloth in a 3mL plastic syringe, minus the needle, and pour the
sample in. Push the liquid through the syringe into a new tube.

4) The phenol/chloroform step uses hazardous chemicals and a hood. So
this may be out of the question for a garage lab. This protocol may
be a good alternative
http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1186
starting from the 5th step (the SDS step). I've never used it but it
looks fairly simple. This will require SDS as well as potassium
acetate. If you live in a city that gets snow, you should be able to
find potassium acetate at a store that sells de-icing supplies.
http://www.senecamineral.com/potassiumacetate.htm

If any of the steps here are unacceptable (too expensive, too toxic,
etc) just tell me and I can try to figure out a suitable alternative.


-Cory

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