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PLANT DNA EXTRACTION by using CTAB Method

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PLANT DNA EXTRACTION by using CTAB Method

Postby veniesh » Sun Sep 27, 2009 5:41 pm

Isolation of total genomic DNA from leaves of plant by using CTAB Method
:shock:
I am doing extraction of DNA by using CTAB method. The first step in this method is freeze the tissue rapidly in liquid nitrogen and grind to a powder with mortar and pestle. My question is can do this DNA extraction without liquid nitrogen? What is the function of liquid nitrogen? Can be use this extraction product for PCR? If cannot, please give the reason and effect? :?: Thank for you answer! :wink:
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Postby JackBean » Sun Sep 27, 2009 5:55 pm

Hi :)

the nitrogen is needed to crush the cell walls and all the cells. It's quite hard to break the plant cell wall, so you do not have many possibilities ;)
If will you have the DNA clean enough (A260/A280 > 1.7), why not to use it for PCR? ;)
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Postby veniesh » Sun Sep 27, 2009 7:21 pm

thanks for answer !!
I am doing extraction without liquid nitrogen. I already get clear and single DNA band.. this DNA product I used in PCR for isolation gene… my question is can i used this DNA extraction product (without liquid nitrogen) in PCR or cannot? DNA extraction product (without liquid nitrogen) can effect PCR product?
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Postby JackBean » Mon Sep 28, 2009 8:17 am

Hi,
well, using only the buffer is OK, I think. The nitrogen shall make the material breakable. I think, that it's not so much needed for isolation of DNA, maybe you just won't have so high yield.
But the PCR should not be inflluenced, the other steps are more important for that ;)
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Postby canalon » Mon Sep 28, 2009 5:38 pm

There can be some inhibitors to PCR if you do not have a good purification method. However, in this case the Liquid N2 is just there to improve cell breaking, the purification is done by the CTAB. So it would seem that if your DNA yield is satisfying, you can try your PCR and see what you get. And if it does not work, I would rather try to improve the purification steps.
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