FFPE Tissue DNA Extraction Kit

Product Composition

Storage and Expiry Date

1.      Proteinase K could be stored at room temperature for at least 1 year. For longer storage, we suggest storing proteinase K at 2-8℃.

2.      If other reagents and items are stored at normal temperature (0~30℃), their performance will remain unchanged within two years; if the product is stored at 2~8℃, the validity period of the product can be extended to more than two years (2~8℃ Stored products should be returned to room temperature before use).

Product Description

The FFPE Tissue DNA Kit is suitable for for isolating and purifying total DNA (including genomic DNA, mitochondrial DNA, and possible viral DNA) from 3 to 8 slices 10 µm tissue sections (area less than 250 mm²). After the tissue is lysed with proteinase K, the DNA will be bound to the spin column, and the degraded proteins and PCR inhibitors will be removed by filtration. After the spin column is washed with Buffer WA and Buffer WB, DNA will be eluted with Buffer TE and can be used for various molecular biology experiments.

Equipment and Reagents to Be Supplied by User

1．  Xylene, absolute ethanol

2．  1.5 ml microcentrifuge tubes, pipette, and tips

3．  Disposable gloves and protective equipment and tissues

4．  Microcentrifuge(s) (with rotor for 1.5 ml and 2 ml microcentrifuge tubes)

5．  Water bath and vortex oscillator

6．  Old paraffin tissue samples may require Carrier RNA (Cat. No. 4003101)

Preparation Before Use

1.      If the centrifuge has a refrigeration function, please set the temperature to 25°C.

2.      Set the water bath temperature to 56°C and 90°C, and incubate Buffer AT and Buffer TE to 56°C.

3.      Add absolute ethanol to Buffer WA and Buffer WB according to the instructions on the label of the reagent bottle, and tick the box on the label to mark "Ethanol added".

Protocol

1.       Use a scalpel to remove excess paraffin blocks from the paraffin tissue specimens, and cut the tissue blocks into 5~10 µm slices.

* If the tissue surface is exposed to air, discard the surface 2-3 layers of slices.

2.       Collect 3 to 8 tissue sections into a 1.5 ml centrifuge tube, add 1 ml of xylene, close the lid, and vortex vigorously for 10 seconds to dissolve the paraffin.

3.       Centrifuge at 13,000 rpm for 2 minutes. Aspirate and discard the supernatant, keeping the pellet at the bottom of the tube.

4.       Add 1 ml of absolute ethanol, vortex for a few seconds to suspend the pellet, and centrifuge at 13,000 rpm for 2 minutes.

* Ethanol will wash away residual xylene.

5.       Aspirate and discard the supernatant, keeping the pellet at the bottom of the tube. Open the lid and leave it at room temperature for 10 minutes or until the ethanol evaporates.

6.       Add 180 µl Buffer AT and 20 µl Proteinase K, vortex to mix well.

7.       Incubate at 56°C for 1 hour (or until the tissue is completely lysed). During this period, vortex several times to help the tissue digestion.

* If there is still a small amount of insoluble matter after the incubation, centrifuge the 1.5 ml centrifuge tube at 12,000 rpm for 1 minute, transfer the supernatant to another clean 1.5 ml centrifuge tube, and then follow step 8.

8.       90 ℃ water bath for 1 hour.

* This step is to partially renature some nucleic acids that have been denatured by formaldehyde.

* If there is only one water bath, please take out the centrifuge tube and place it at room temperature. When the water bath reaches 90°C, put the centrifuge tube into the water bath.

9.       Add 200 µl Buffer SL and 200 µl absolute ethanol, and mix by inverting 4 to 6 times. Spin for a few seconds to allow the mixture on the tube cap to settle to the bottom of the tube.

* If DNA is extracted from old paraffin tissue blocks, please add 3 µl Carrier RNA (Cat. No. 4003101) at this step. The DNA in old paraffin tissue samples is very severely degraded. It must be effectively adsorbed to the spin column with the assistance of Carrier RNA.

10.    Pipette the mixture into a spin column (the spin column is placed in a 2 ml collection tube), centrifuge at 12,000 rpm for 30 seconds.

* Be careful not to get the solution on the edge of the spin column mouth, otherwise the spin column will not be cleaned in subsequent washing steps.

11.    Discard the filtrate in the 2 ml collection tube, place the spin column back into the 2 ml collection tube, add 500 µl Buffer WA to the spin column, centrifuge at 12,000 rpm for 30 seconds.

* Ensure that absolute ethanol has been added to Buffer WA.

* The filtrate does not need to be discarded completely. If you want to avoid contamination of the centrifuge by the filtrate adhering to the mouth of the centrifuge tube, you can place the 2 ml collection tube upside down on a paper towel and tap it once.

12.    Discard the filtrate in the 2 ml collection tube, place the spin column back into the 2 ml collection tube, add 600 µl Buffer WB to the spin column, centrifuge at 12000 rpm for 30 seconds.

* Ensure that absolute ethanol has been added to Buffer WB.

13.    Discard the filtrate in the 2 ml collection tube, place the spin column back into the 2 ml collection tube, and centrifuge at 14,000 rpm for 1 minute.

* If the centrifuge speed cannot reach 14000 rpm, centrifuge at full speed for 2 minutes.

* Do not pass over this step, otherwise the purified nucleic acid may be mixed with ethanol, which may affect subsequent PCR results.

14.    Discard the 2 ml collection tube, place the spin column in a clean 1.5 ml centrifuge tube, add 60~100 µl Buffer TE incubated at 56°C to the spin column, and let stand at room temperature for 1 minute, centrifuge at 12000 rpm for 30 seconds.

* If the centrifuge does not have a leak-proof cover, please change the centrifugation conditions to 8000 rpm for 1 minute to prevent the 1.5 ml centrifuge tube lid from falling off and damaging the centrifuge.

15.    Discard the spin column, and the eluted DNA can be used immediately for various molecular biology experiments; or the DNA can be stored at -20°C for later use.