Spin FFPE DNA Kit-HYC241
After the digested sample is incubated at a higher temperature, it is cross-linked with formalin that removes free DNA, which effectively improves the yield and purity of DNA; the optimized buffer system enables the DNA in the lysate to be specifically bound to the adsorption membrane. High-purity DNA can be obtained by effectively removing inhibitors through a two-step rinsing step, followed by final elution with low-salt buffer or water. At the same time, it is equipped with a high-efficiency micro-adsorption column, and the elution volume can be as low as 20 μL. The purified DNA can be used directly for PCR, Real-time PCR, SNP genotyping, STR genotyping, next-generation sequencing and pharmacogenomics research. The molecular weight of DNA isolated from formalin-fixed, paraffin-embedded samples is generally lower than that from fresh or frozen samples. The degree of DNA fragmentation depends on the sample type, storage time, and fixation conditions.
Components
|
Components |
50T |
|
Buffer DS |
30 mL |
|
Buffer GTL |
15 mL |
|
Buffer GL |
15 mL |
|
Buffer GW1* |
13 mL |
|
Buffer GW2* |
15 mL |
|
Buffer TE |
10 mL |
|
RNase A (100 mg/mL) |
0.4 mL |
|
Proteinase K |
2×1.25 mL |
|
Spin Columns DF with Collection Tubes |
50 |
|
Centrifuge Tubes (1.5 mL) |
50 |
Storage
The Spin Columns DF can be stored at 2-8°C. Other components can be stored at room
temperature (15-30°C) .
Required Reagents and Equipment
- Absolute Ethanol
Instructions
Preparations
- After obtaining the sample, fix the sample in 4%-10% formalin as soon as possible, and the fixation time should be 14-24 hours. If the time is too long, the genome will be broken and the downstream experiments will be affected. If the formaldehyde fixation time is too long or the sample is stored for a long time (>1 year), the integrity of the DNA is easily damaged, and long fragments cannot be amplified.
- Make sure that the sample before embedding is completely dehydrated, and the residual formalin will inhibit the effect of Proteinase K.
- Before the first use, add 100% ethanol to Buffer GW1 and Buffer GW2 according to the instructions on the reagent bottle label.
- Before use, please check whether Buffer GTL, Buffer GL and Buffer DS are crystallized or precipitated. If there is crystallisation or precipitation, please re-dissolve Buffer GTL, Buffer GL and Buffer DS in a 56°C water bath.
- Before starting the experiment, preheat the water bath or constant temperature mixer to 56°C, and the centrifuge to 25°C.
- If downstream experiments need to reduce the low frequency C>T | G>A transition (artificial mutation) to minimize the risk of false positives, 7 μL UNG (1U/uL)can be added after incubation at 90°C for 1 hour. ).
Protocol
- Sample processing
- Paraffin-embedded samples: Trim off the excess paraffin in the tissue block with a scalpel to expose the tissue and cut into 5-10 μm thin slices. Take about 1×1 cm2 slices (about 4-5 slices in total) and put them in a centrifuge tube (self-provided), add 160 μL BufferDS, vortex for 10 seconds, then add 180 μL Buffer GTL and 20 μL Proteinase K, vortex and shake for 10 seconds. Centrifuge at 12,000 rpm, 25°C for 1 minute.
Note: 1) If the surface of the sample has been exposed to the air, please discard the 2-3 pieces exposed to the air. 2) DS will solidify below 18°C, if solidification does not affect the following experiments.
- Samples in formalin fixative: take 20 mg samples, cut them into small pieces, placethem in a centrifuge tube, add 500 μL of 10 mM PBS (pH 7.4), vortex and centrifuge at 12,000 rpm for 1 minute. Discard the supernatant and repeat 3 times. Add 180 μL Buffer GTL, 20 μL Proteinase K and vortex to mix.
- Incubate at 56°C for 1 hour until the sample is completely dissolved. Incubate at 90°C for1 hour. Centrifuge at 12,000 rpm at 25°C for 1 minute. Use a pipette to carefully pipettethe lower aqueous phase (about 180 μL) along the tube wall into a new centrifuge tube, avoiding the suction of the sediment at the bottom of the tube and the upper wax liquid.
Note:1) The sample incubated at 56°C can be placed at room temperature until the temperature of water bath or dry bath reaches 90°C before incubating at 90°C. 2) Optional step: Add 7 μL UNG (1U/μL) , 50°C, 5 min, no concussion.
- Optional step: To remove RNA, lower the temperature of the sample to room temperature, add 2 μL of RNase A (100 mg/mL), shake and mix well, and leave at room temperature for 2 minutes.
- Add 20 μL Proteinase K, incubate at 65°C, 450 rpm for 15 min.
- Add 200 μL of Buffer GL, vortex to mix, then add 200 μL of 100% ethanol, and vortex to mix thoroughly. Briefly centrifuge to collect the solution on the tube wall to the bottom of the tube.
Note: 1) Mix well immediately after adding Buffer GL and 100% ethanol. 2) After adding Buffer GL and 100% ethanol, a white precipitate may occur, which will not affect subsequent experiments. 3) If you need to operate on multiple samples, you can mix Buffer GL and 100% ethanol before adding the samples.