Question
Asked 24th Feb, 2015
  • Tomsk National Research Medical Center

Is it possible to get DNA from gDNA eliminator spin columns (Qiagen)?

Hi!
I isolate tumor cells from breast tumor sections by laser microdissection and extract RNA by RNeasy Plus Micro Kit (Qiagen). But I would like to also get DNA from the same microdissected samples. For example, can I get DNA from gDNA eliminator spin columns, which are used to eliminate DNA from RNA samples in RNeasy Plus Micro Kit? Is it possible to elute DNA from these columns? Will such DNA be good for further molecular analyses?
Thank you in advance!

Most recent answer

24th Dec, 2017
Morteza Zahedi
Academic Center for Education, Culture and Research
Hi Evgeny,
this topic is very old, but do you remember the results?
did you isolate DNA from gDNA eliminator column?
best

Popular answers (1)

25th Feb, 2015
Cristian Köpfli
University of Notre Dame
Hi Evgeny,
It is possible, I have used the following protocol. I don't think the DNA quality is as good as with a specific DNA extraction kit (I have only processed few samples), but better than nothing. As far as I know Trizol is very good for RNA, but not for DNA (e.g. hard to do Illumina DNAseq on samples extracted from Trizol).
This is the protocol:
Place gDNA elimination column on a washed S-Block, add 500µl AW1 Buffer (cat. No. 19081) to each well, seal with tape sheet and centrifuge at 6000rpm (5600g) for 2min.
Discard flow-through, add 500µl AW2 Buffer (cat. No. 19072) to each well, seal with tape sheet and centrifuge at 6000rpm (5600g) for 4min.
Discard flow-through, centrifuge gDNA elimination column for 4min at 6000rpm (5600g) with empty S-block to dry themembrane.
Place the columns on top of a 96-well master plate (Sarstedt, 72.1980.202): DNAplate_#_date
Add 50µl pre-warmed (~40°C) AE Buffer (cat. No. 19077) to the columns, incubate for approx. 30min, centrifuge at 6000rpm (5600g) for 4 min.
repeat step 5 to get 100µl coextracted DNA for rescuing some difficult genotyping samples.
7 Recommendations

All Answers (6)

25th Feb, 2015
Ricardo Frausto
UCLA
Evgeny, probably best to contact Qiagen directly to get a fast and accurate answer to your question. That being said, have you considered using Trizol/Tri-Reagent? You get both RNA and DNA (and protein if you are keen). 
25th Feb, 2015
Evgeny V. Denisov
Tomsk National Research Medical Center
Thank you, Ricardo! I tried Trizol, but it works no so good with small samples (300-1000 cells).
25th Feb, 2015
Cristian Köpfli
University of Notre Dame
Hi Evgeny,
It is possible, I have used the following protocol. I don't think the DNA quality is as good as with a specific DNA extraction kit (I have only processed few samples), but better than nothing. As far as I know Trizol is very good for RNA, but not for DNA (e.g. hard to do Illumina DNAseq on samples extracted from Trizol).
This is the protocol:
Place gDNA elimination column on a washed S-Block, add 500µl AW1 Buffer (cat. No. 19081) to each well, seal with tape sheet and centrifuge at 6000rpm (5600g) for 2min.
Discard flow-through, add 500µl AW2 Buffer (cat. No. 19072) to each well, seal with tape sheet and centrifuge at 6000rpm (5600g) for 4min.
Discard flow-through, centrifuge gDNA elimination column for 4min at 6000rpm (5600g) with empty S-block to dry themembrane.
Place the columns on top of a 96-well master plate (Sarstedt, 72.1980.202): DNAplate_#_date
Add 50µl pre-warmed (~40°C) AE Buffer (cat. No. 19077) to the columns, incubate for approx. 30min, centrifuge at 6000rpm (5600g) for 4 min.
repeat step 5 to get 100µl coextracted DNA for rescuing some difficult genotyping samples.
7 Recommendations
25th Feb, 2015
Evgeny V. Denisov
Tomsk National Research Medical Center
Many thanks, Cristian, for the protocol! I will try it surely. But I think that DNA samples should be additionally treated with proteinase K to remove proteins with whom DNA can be bound.
24th Dec, 2017
Morteza Zahedi
Academic Center for Education, Culture and Research
Hi Evgeny,
this topic is very old, but do you remember the results?
did you isolate DNA from gDNA eliminator column?
best

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I have a question to Jochen Wilhelm and other experts in the field. I am a physician scientist currently working in basic science. We work on rodent experimental models and use qPCR a lot. We follow very stringent qPCR protocols with various reference genes for different tissues and cell lines. I believe our technical standards are good and our results are controlled for as many confounders as possible.
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