Tag Archives: DNA extractions

Quick update: 96-well plate CTAB DNA extraction with fresh tissue

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When I posted the protocol I have been using for CTAB DNA extraction in 96-well plates, I included results from a few plates I did starting from dried H. anomalus leaves I collected a couple of months earlier in Utah. While they showed that the method worked well enough when starting from “difficult” material, they were not exactly what you’d dream of when you decide to extract DNA, especially if you are starting instead from fresh material.

Here are the results from a plate of extraction I did starting from individual small (1.5-2 cm in length), young leaves from ~3 month-old H. anomalus plants. I collected the leaves directly in 96-well plates (I already put one metal bead in each well), put them on dry ice until I got to the lab (a couple of hours), left them overnight in the -80, and started extracting DNA the day after.

DNAExtractionF2Plate1

The final volume was 50 microliters, so total yield is for most samples between 10 and 30 micrograms of DNA. These are “real” DNA concentration measured by Qubit. Both average yield and purity are considerably higher than for dry tissue, and they are comparable to what you would get starting with frozen tissue using the single tube protocol (but you save a ton of time). Hope this gets you all more thrilled about 96-well plate DNA extractions 🙂

96-well plates CTAB DNA extraction

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When I was working with Arabidopsis, 96-well CTAB DNA extraction was my best friend, and I spent many days extracting away tens of thousands of samples. Good times.

DNA extraction is much less pleasant in sunflower, but since I was reasonably happy with the results of single-tube 3% CTAB DNA extractions, I though I would try to scale it up to a 96-well plate format. Results of earlier attempts, with the participation of Brook and Cris, ranged from inconsistent to disastrous. Things all but improved when I tried again after coming back from Utah with a few hundreds dried samples. Since though the prospect of extracting them all one by one didn’t sound very attractive, I put some more effort into improving the protocol, and now it works quite nicely.

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Plant DNAzol

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Hi all,

Here’s another method for DNA extraction.  The blog is stuffed to the gills with DNA extraction methods.  The current standards are ‘Qiagen-like’ columnless – used to generate the DNA for the genome sequencing project and CTAB.  I add this protocol because itis
easy and it is effective.

I extracted from Ha89 and harvested ~ 115 ng/uL from 20 cm tall plants.  I also purposely ‘took it slow,’ letting tissue thaw after freezing to see PlantDNAzol’s efficacy.  It’s efficient.

PlantDNAzol-uncut

Quality is good

The gel to the left:

Right most lane contains Ha89 genomic DNA – 10 uL loaded of 70 at 115 ng/uL DNA – 260/280 was 1.78.  260/230 was ~1.00 (I suspect I could have added an additional ethanol wash to remove Guandine from the DNA mixture.

Is the DNA useful?  Can downstream reactions proceed?

Yes.  I digested the DNA with a methylation sensitive restriction enzyme, PstI and a methylation insensitive enzyme, EcoRV

PlantDNAzolcut-22hrs

<–The gel to the left :

Leftmost lane  Ladder

A vector digested with PstI,

Ha89 gDNA digested with PstI 240 minutes,

Ha89 gDNA digested with EcoRV 240 minutes.

The DNA digests.  But does it contain contaminants that upset the enzymes over long incubations?  Overnight?

PlantDNAzolcut-240minutes

Gel above:  From left- Ladder, unrelated vector digest,

Ha89 gDNA digested with PstI 22 hours

Ha89 gDNA digested with EcoRV 22 hours.

Protocol for DNAzol extraction (exactly as published by LifeTech but easier to follow – http://tools.lifetechnologies.com/content/sfs/manuals/10978.pdf:

Have these items on hand:

1 0.6 mL DNAzol per 100 mg sample

2. 0.3 mL chloroform per 100 mg sample

3. Timer

4. 100% ethanol (0.225 mL per sample),

5. 75% ethanol (0.3 mL per sample)

Handle all inversions carefully.  When you see invert or shake handle your samples gently

1. Mix 100 mg ground tissue with 0.3 mL PlantDNAzol – 100 mg is max.  Overdoing will hurt your yield.

2. Invert gently to aid in lysis and dispersion

3. Once completely dispersed incubate at RT, 5 min, shake periodically.

4. Add 0.3 mL chloroform and mix.

5. Once completely dispersed incubate at RT, 5 min, shake periodically.

6. Centrifuge at RT, 12 000 g (NOT rpm) 10 min

7. Harvest the supernatant. – you’ll see a phenol/chloroform styled triple layer.  The middle layer will be pulpy containing your cellulosic debris and proteins.  Don’t collect the middle layer.  Less is more

8. Mix supernatant from 7 with 225 uL 100% ETOH.

9. Incubate at RT, 5 min

10. Centrifuge mixture 5000 g 4 min – get preparing for step 11

11. Make a PlantDNAzol – Ethanol mixture:  For one sample mix 0.3 mL PlantDNAzol with 0.225 mL 100% ethanol.

12. Discard the supernatant from 10 and mix it with 0.3 mL of the mixture prepared in step number 11

13. Incubate as in step 9.

14. Spin as in step 10.

15. Pour off supernatant

16 Wash pellet with 75% ethanol – 0.3 mL – this step can be repeated if your 260/230 isn’t adequate.  Guanidine absorbs strongly in 230 nm wavelength

EDIT: repeat step 16 for a total of 2 washes.

17. Spin as in step 10.

18. Remove supernatant – if your samples are green repeat step 16.

19. Resolubilize your DNA in TE or NaOH.  Make sure to run your bead of TE over the wall of the tube to collect your DNA.

 

EDIT – Less is more as is usually the case with DNA extraction.  I harvested from 38, 60, and 100 mg of tissue.  – A sweet spot for tissue quantity is 45 to 60 mg for the given amount of Plant DNAzol

Qubit quantifiication:  45 mg of tissue yielded 264 ng/uL ug/mL.  60 mg, 305 ng/uL.  100 mg, 12.4 ng/uL

How my PhD was saved by DTT, or: how to get moderate quantities of clean, unsheared DNA from tricky tissues

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I have been trying for months to find a high-throughput solution to DNA extraction from lyophilized (freeze-dried) leaf discs of Helianthus argophyllus, a species known for its intransigent polyphenolics and low DNA yields. And I appear to have found one, thanks to Horne et al. 2004 (here) and Dow Chemical!

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Ph*cking phenolics!

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Hi guys,

I have extracted sunflower DNA a dozen times now.  I get one consistent problem a gelatinous film coprecipitating with my DNA (carbohydrates/polysaccharaides).  I also get sharp smelling phenolic compounds.  These occur regardless of DTT/2-BME addition.

I found a paper detailing the use of 2-butoxyethanol (2-BE) to remove strawberry polysaccharides and phenolics.  The researcher’s idea is to perform a two-step precipitation:

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Momma’s CTAB

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Like a few other people in the lab, I’ve been struggling with DNA extractions of late. I’ve tried several methods, including the “columnless” method that a few people are using. But I was having pretty spotty luck. Then, during a bout of methodological soul-searching, I also tried a CTAB protocol that used to be my go-to method. It failed me a few weeks ago, but in a second trial last week, it delivered the goods: loads of very pure, high molecular weight DNA. It might not work for all plants, and there do appear to be situations in which it is not the best option, but if anyone wants to get back to down-home, from-scratch extraction, the way momma used to do it, then give this one a try:

Please note: the results below are for Streptanthus (the plant I’m studying; Brassicaceae); I’m trying it for some Asteraceae right now, and will update the post once I have results.

Momma’s DNA Isolation via CTAB

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