Quick-DNA™ FFPE Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
If the tissue sample is too large for the digestion volume, scale up the digestion to 200 µl while keeping the amount of Proteinase K the same.		- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Upstream tips
If the tissue sample is too large for the digestion volume, scale up the digestion to 200 µl while keeping the amount of Proteinase K the same.
Downstream tips
- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Publication protocol

DNA retrieval from freshly frozen mouse tissue
The tissues were thawed in a water bath at 4°C, and then immediately homogenized in 50mM Tris–HCl buffer (pH 8.0) containing 10mM ethylenediaminetetraacetic acid (TE buffer, 3 ml per gram tissue) with a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 3000g for 10min at 4°C. The genomic DNA in the nuclear pellet (equivalent of 50mg of wet tissue weight) was isolated by CHCl3/phenol extraction as described (9,16).

Deparaffinization, rehydration and isolation of DNA from mouse FFPE tissues and human kidney tissue sections
The mouse liver or kidney were dissected from the paraffin block, and the bulk of the paraffin removed with a scalpel. The tissues were submerged in p-xylene, and then underwent rehydration, followed by homogenization, and centrifugation at 3000g (9). The nuclear pellet, using the equivalent of 20−25mg of dry weight of deparaffinized tissue, was processed with the ZR FFPE DNA MiniPrep kit (Zymo Research) according to the manufacturer’s instructions with minor modifications (9). For human kidney, two 10 μm sections, each with a surface area of 0.9−1.8cm2, were placed in an Eppendorf tube and washed with p-xylene (1.5 ml), followed by ethanol/water (95/5%). The tissue was air-dried for 30min and then resuspended in TE buffer (0.1 ml). The samples were digested with proteinase K for 4h at 55°C in lysis buffer. A complete description of the retrieval of DNA from FFPE tissue is provided as Supplementary Protocol S1, available at Carcinogenesis Online. To estimate the size distribution of the recovered DNA, 250ng of each DNA was electrophoresed through a 1% agarose gel in 1× TAE buffer and 0.5 µg/ml ethidium bromide. Each gel contained multiple lanes loaded with a 100bp ladder (New England BioLabs, Beverly, MA). The gels were photographed and staining intensity in each lane measured with ImageJ software. The molecular weight of DNA was estimated from standard curves constructed from the migration of authentic standards.

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Manufacturer protocol

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