Problems with DNA Extraction of Tomatoes - DNA spooled is not clear and translucent (Apr/09/2006 )
The problem: When I perform DNA extraction on tomatoes, the DNA spooled shows signs of reddish pigments on it. This becomes hard when teaching children that DNA is suppose to be colourless. I have been told that incorporating meat tenderizer could remove the this pigments on the DNA, however, I am not sure of the concentration and the volume to add. I have been experimenting with different ways to incorporate meat tenderizer into the protocol. I have tried adding to the DNA extraction buffer and I have also tried adding it into the tomato extract after cheesecloth filtration.
So could anyone suggest to me a simple yet effective way of eliminating this pigmentation from my DNA using a combination of DNA extraction buffer with meat tenderizer.
DNA extraction buffer components:
1M Tris-HCl pH 7.5
Thanks in advance
Switch to onions.
I have thought about switching to onions, but as this is an enrichment programme for the kids, I dun wan to see them tearing while they are mashing their onions, lol. Meat tenderizer contains protease, so could that really eliminate the reddish pigments Thanks phage for the quick reply.
You could eliminate the pigment by doing organic extraction with either mixture of phenol:chloroform or only chloroform, but that's not recommended since childrens are doing it.
I'd prefer explaining to them that the "crude DNA" contains RNA, residual proteins, pigments (esp. lycopene) and also a bulk of DNA. To obtain a purified DNA, further purification steps are required, such as RNase treatment, organic extraction and ethanol precipitation and wash in order to get a purified grade for PCR etc... BUT the "crude DNA" is suffice to demonstrate how DNA looks like and IF you've got agar and electrophoresis set, you could separate it and let them visualize it with non-carcinogenic dye (such as crystal violet Ref: http://www-biology.ucsd.edu/labs/aroian/protocols/pepsi.htm).
That'd cheer them up.
look at > http://gslc.genetics.utah.edu/units/activities/extraction/ <
give a nice example for kids....But I never tried this way, but looks good.
we prepared kiwi-DNA and it was not green!
we only used a mix of water and detergent that you use to do the dishes and let the kiwi-pieces inthere for 15 min at 65C. Pass the mixture throug a coffee-filter, add some ETOH and there you have wonderfull threads of DNA! Very children-friendly, cheap and not dangerous.
Maybe I should try this protocol to prepare my Sesbania-gDNA...
I've done DTAB/CTAB genomic DNA extractions on a variety of plants. I always follow up with PCI/EtOH precipitation, but I could not get rid of the pigments! Some plants produced purple-ish tints, others green-ish, and others perfectly translucent.
I have yet to figure out a further purification method that will remove these pigments, but my downstream applications were never affected. I can see how you'd like the kids to learn how things are supposed to turn out, but maybe an important lesson is that sometimes science produces unexpected results. These peculiar results are what pushes science forward towards discovery and innovation.
Eh... at the very least, it's a good excuse