Hi everyone,
I'm now working on DNA and RNA extraction for FFPE samples using "RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE" (Life Technologies).
After digestion by Proteinase K (usually incubate more than 24-48hours until all samples were transparent), I followed the manufacturer's protocol: I added mixture of Isolation additive, Ethanol with Digestion products and pippetted the mixture on the Filter. As normal, the mixture will not pass through the filter. However, some of tubes the mixture passed through the Filter (about 50-100 mcL) prior to centrifuging.
Can anyone explain why this phenomenon happen and how to solve it?
Thanks a lot
If that doesn't affect your DNA quantity, we shouldn't be bothered by the phenomenon. Because I have had this and I didn't have problem with the DNA quantity. DNA will bind to the membrane during the cleaning step, no worry.
Do you mean the filter in a centrifugation column? If so, that is normal. Gravity is always working on the liquid, always making it fall. It simply goes slower because of adhesive forces of liquid while sliding past so much surface area inside the filter. The speed varies by the solution. It is a race between the adhesive forces and the force of gravity. Centrifugation simply multiplies the gravitational force. And the reason your DNA is safe is because the adhesive forces can resist even that increased gravitational force until you apply elution solution.
@Hung King Tiong: Thanks for your kind answer. Yes I'm worried that it would affect the genomic material quality. Because I have performed DNA or RNA extraction for many samples and this is the first time I see it.
@Michael Gines: yes, I mean the Filter catridge in centrifuge tube. As I mentioned above this is my first time so I'm a bit worried about the DNA quality ;)
Additionally I have another question regarding proteinase K. Some samples did not clarify after 48h digestion and incubation with proteinase K. According to the manufacturer the ideal amount of proteinase K used for digestion step is 4mcL. Can I increase the amount to 8mcL for those undigested samples? Will it affect the quality of genomic material?
Thanks
If this method gives insufficient dna quantity for your downstream reaction, pls consider using the bead method for dna extraction. It has been working incredibly well for dna extraction of both Gram types bacteria. It even gave 100% successful dna extraction of listeria monocytogenes which bax protease sometimes didn't work.
The link of the bead extraction method :
http://www.sciencedirect.com/science/article/pii/S0167701205001077
I have used up to 10% Proteinase K (20 µL per 200 µL reaction) in digestion of fresh or frozen tissues. Generally these were incubated overnight at 55° C. What temperature are you incubating at?
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