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.
--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?
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