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genomic DNA extraction from formalin fixed tissues (not embedded) for genotyping

gDNA formalin fixed PCR extraction

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#1 Roo

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Posted 13 July 2012 - 07:38 AM

Does anyone have a good protocol for isolating genomic DNA from formalin fixed tissues, preferably without using a kit? I need to repeat genotyping of some mice that are now stored in formalin. I understand that long term storage in formalin can greatly decrease my success rate, but I don't have fresh or frozen tissue to use so I have no choice.

I found protocols using kits, salt precipitation, and various digest buffers, but most are for paraffin embedded samples. I don't have to do a xylene step to get rid of the paraffin, so I'm thinking I could try picking a protocol and starting after the xylene step. However, I don't know if I should perform wash steps and/or the drying steps that are found in so many protocols. I may just try something since I don't have anything to lose, but I was hoping somebody might have a tried and true method that usually works for their lab.

Any information or suggestions would be greatly appreciated!

-Roo

Edited by Roo, 13 July 2012 - 07:39 AM.


#2 Adrian K

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Posted 16 July 2012 - 07:29 AM

There is one article:
Analytical Biochemistry 395 (2009) 265–267 doi:10.1016/j.ab.2009.08.016

This protocol using mineral oil, not xylene. See if this paper helps.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#3 Roo

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Posted 16 July 2012 - 09:18 AM

Thanks, but this article describes the use of mineral oil as a deparaffinization agent. It was used in conjunction with commercial DNA extraction kits. My samples aren't embedded in paraffin. They're just mouse carcasses sitting in formalin. For the time being, I can't justify buying a DNA extraction kit for just a few samples when I already have most things needed for extraction. I was simply looking for a protocol that someone has personally used. Thanks for trying though! I will probably end up doing a bit of trial and error. If it works, I'll post it!

#4 Adrian K

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Posted 17 July 2012 - 04:32 AM

I'm sorry as I overlooked your query... my bad. However, based on Google search, here are 2 papers which might help you.

1) This protocol uses Tetramethylsilane (TMS)-Chelex (not sure if you have this chemical in your lab)
http://eprints.soton...SciMar_2010.pdf

2) This one looks more promising, use normal chemicals (DNA extraction from formalin fixed Dolphin tissues):
http://www.zoo.ba.go..._extraction.pdf
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#5 Roo

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Posted 17 July 2012 - 07:22 AM

Thanks Adrian! The second article is perfect!!! It's similar to others that I found, but better. I probably won't do it this week, but I'll let you know how it turns out. Thanks again!

-Roo : )





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