kitchen DNA extraction not showing on the gels

[Tom Hodder]

Hi all,

I've tried this procedure a few times, and as yet I've not been able to see any bands in the samples channels that we extracted from the tomatoes.

Any suggestions on what I am doing wrong, because I'm starting to pull my hair out ;-)

Basically we have done a "kitchen" extraction of DNA from tomatoes, and we are getting some white gooey precipitate and a kind of red-ish pellet forming and rising to the top after the application of the cold isopropanol.

However after redissolving this in TE buffer, there is nothing showing in the channel when run on a gel. (The ladder lane is showing bands nicely. Also previously extracted DNA using a kit is showing in a positive control lane)

Here is the link to the google doc with the protocol and the stages photo;
https://docs.google.com/document/d/1mXuL7l6o13a20EKw2KaahHs1RR04KdYhqwDXSU2oQgo/edit?usp=sharing

Extraction of DNA from tomatoes for running in 2% agarose/EtBr gel

86 g tomato

100 mL deionized water

5 mL detergent

25g salt

12g Sodium citrate

ice cold isopropanol

5 mL TE buffer

loading dye

ladder

extraction

1) chop and pulp tomato and put in sandwich bag

2) add 100 mL deionized water, 5 mL detergent, 25g salt to bag

3) squash and mix tomato in bag and leave for 20 mins

4) strain liquid through gauze into test tube, fill to about ¼



5) gently fill the test tube to ¾ full with ice cold isopropanol and leave for 15 minutes



6) tried a few variations here; (none of which worked)

i) isolate the pellet of red precipitate that floated to the top

redissolve in TE buffer and add 1/5 loading dye to make a 1/6 dilution

ii) spin down a sample from the white cloudy stuff that has formed at the top of the alcohol layer

redissolve in TE buffer and add 1/5 loading dye to make a 1/6 dilution



7) run the extracted DNA with loading dye;



8) we are seeing the ladder lanes showing up in the UV, and the loading dye is running, but the sample lanes are completely empty.

There is a very faint suggestion of a glow in the well, but hardly anything

What are we doing wrong?

[Dakota Hamill]

Maybe I missed it but, what DNA stain are you using? gel green?

You're extracting genomic DNA with probably a lot of contamination, and who knows how much of it.  If anything, you should only see a glow in the wells, and depending on how much of it there is and how chopped up it is, maybe a smear below the wells.    If I repeated the experiment I'd consider it a success if I saw a glow in the wells as that's where the massive pieces of DNA you extract should be, probably still in tight cahoots with some histones or maybe other proteins that don't let the DNA dye get close enough to bind efficiently for a good UV response.

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[Mac Davis]

How can the experiment be improved?

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[Tom Hodder]

On 21 April 2014 00:56, Dakota Hamill <dko...@gmail.com> wrote:

Maybe I missed it but, what DNA stain are you using? gel green?

Ethidium Bromide

You're extracting genomic DNA with probably a lot of contamination, and who knows how much of it.  If anything, you should only see a glow in the wells, and depending on how much of it there is and how chopped up it is, maybe a smear below the wells.    If I repeated the experiment I'd consider it a success if I saw a glow in the wells as that's where the massive pieces of DNA you extract should be, probably still in tight cahoots with some histones or maybe other proteins that don't let the DNA dye get close enough to bind efficiently for a good UV response.

This was the state of the current gel. It has 2 ladders and the rest are various extracted potions. You can see that the wells have some UV response.

 

[Nathan McCorkle]

The incubation times seem too long, nucleases could be chewing up the DNA in that time, if you're seeing a smear on the gel. I recommend using a coffee filter rather than gauze, or even doing it in two stages (gauze then coffee filter)... and your salts/ions seem much too massive... I'd think less than a gram in total should be fine. Many protocols I just found on Google are using 25-50g salts per liter, if they're included at all.

You probably also want to dry the DNA in an oven before redissolving.

You also seem to be using a lot of detergent, from my practice I've used just a drop or whatever comes out of the bottle before I can stop squeezing.

After drying the DNA, you could do an alcohol rinse, but you'll need a centrifuge to get the pellet so it doesn't get decanted out with the rinse alcohol. The detergent (if ionic) and loads of salts could definitely be messing with your results.

The picture in the next email you sent doesn't look like fluorescense, rather it looks like absorbance... so your extraction might just not be pure enough in general. If the genomic DNA isn't too chewed up (by nucleases or mechanical shearing), it won't migrate very far anyway, certainly much much less than a 1kb ladder would.

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[Tom Hodder]

Well, after rerunning with reduced NaCl and detergent, and being much more careful with the re-suspension in TE, there is more definitely glowing in the sample wells;

I'll updated the protocol, and try and run again with a longer gel time and maybe more +V

Cheers,
Tom

On 21 April 2014 02:03, Nathan McCorkle <nmz...@gmail.com> wrote:

The incubation times seem too long, nucleases could be chewing up the DNA in that time, if you're seeing a smear on the gel. I recommend using a coffee filter rather than gauze, or even doing it in two stages (gauze then coffee filter)... and your salts/ions seem much too massive... I'd think less than a gram in total should be fine. Many protocols I just found on Google are using 25-50g salts per liter, if they're included at all

 

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