Paraffin-embedded Tissue Genomic DNA Extraction Kit, 50 T (for Nucleic Acid Extraction)

Product Information

Description/Introduction

The kit is suitable for genomic DNA extraction of formalin fixed and paraffin embedded tissue (FFPE), using special dewaxing reagent, no xylene and other organic solvents, safe and non-toxic, can effectively remove paraffin and release tissue samples. The kit can quickly and effectively extract genomic DNA from paraffin-embedded tissues by using special lysis buffer combined with centrifuge column method. The extraction process can be completed within 1 hour, and DNA has high yield, high purity and integrity, so it is suitable for a variety of downstream molecular biology experiments, such as PCR, Real-Time PCR, SNP genotyping and other molecular biology experiments.

Storage and Handling Conditions

RNase A and Proteinase K are shipped with wet ice and stored at -20°C. Other reagents are shipped and stored at room temperature; valid for 12 months.

Product Contents

Before starting (please read carefully)

1. Prepare water or metal baths at 80°C, 56°C and 90°C in advance.

2. If Buffer GL and Buffer GB precipitates, please heat at 65°C to dissolve and use it after returning to room temperature.

3. Please add 21 mL anhydrous ethanol to Buffer GP and 56 mL anhydrous ethanol to Buffer PW before first use, mix it thoroughly and then use it.

Assay Protocol / Procedures

1. Samples preparation:

a. Paraffin sections: scrape 5~8 paraffin sections (1×1 cm2 size) with a sterilized scalpel and collect tissue fragments and place them in a 1.5 mL centrifuge tube.

b. Paraffin blocks: scrape the tissue sample of about 30 mg with a sterilized scalpel, remove the excess paraffin as much as possible and cut up the sample.

c. Sample tissue soaked in formalin: dry the liquid on the sample surface with filter paper, take about 30 mg sample, cut it up and place it in 1.5 mL centrifuge tube, add 500 μL PBS buffer (pH 7.4), vortex oscillation for 10 s, then centrifuge at 12,000 rpm for 1 min at room temperature, discard the supernatant, repeat 3 times.

2. Transfer the sample to a 1.5 mL centrifuge tube, add 500 μL Buffer DP, incubate at 80°C for 3 min and vortex oscillation for 10 s while the sample is hot.

3. Add 200 μL Buffer GL to the centrifuge tube, mix thoroughly with vortex, centrifuge at 12,000 rpm for 1 min at room temperature, and form two layers of solution (upper layer is oil phase, lower layer is water phase).

4. Add 20 μL Proteinase K and 10 μL RNase A to the lower aqueous phase, gently mix with a pipette (try not to destroy the stratification), incubate at 56°C for 20 min.

5. Then transfer the centrifuge tube to a 90°C heating block, incubate for 10 min (if there are more tissue blocks that are not digested completely, the incubation time can be appropriately extended to 20 min), and cool to room temperature.

6. Add 200 μL Buffer GB and 200 μL anhydrous ethanol to the sample in the previous step, and mix well by vortex oscillation.

7. Centrifuge at 12,000 rpm for 1 min, and form two layers of solution (upper layer is oil phase, lower layer is water phase).

8. Remove the aqueous solution from the lower layer into DNA Spin Column (do not get the upper oil phase solution and other impurities). Centrifuge at 12,000 rpm for 2 min at room temperature, and discarded the filtrate.

9. Add 500 μL Buffer GP to DNA Spin Column, centrifuge at 12,000 rpm for 1 min at room temperature, then discard the filtrate.

10. Add 600 μL Buffer PW to DNA Spin Column (please add Buffer PW along the pipe wall to help flush the residual salt on the pipe wall), centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate.

11. Repeat step 10.

12. Place DNA Spin Column on the collection tube, centrifuge at 12,000 rpm for 2 min. Transfer Spin Column to a new Nuclease-free 1.5 mL centrifuge tube.

13. Open the lid of DNA Spin Column, leave it at room temperature for 3~5 minutes to make the ethanol evaporate completely.

14. Add 50~100 μL Buffer TE or Nuclease-free Water preheated at 65°C to the center of the DNA Spin Column membrane, stand at room temperature for 5 min. Centrifuge at 12,000 rpm for 2 min to collect DNA. To get a higher concentration of DNA, you can also add the first eluent back to DNA Spin Column, then stand at room temperature for 5 min and centrifuge at 12,000 rpm for 2 min to elute DNA again.

Note

1. Please read the Product Manual carefully before use.

2. The yield and integrity of DNA extracted by this kit depend on the type of sample, fixed time, fixed condition and sample storage time. The fixed time is preferably within 8~24 hours. If the fixed time of the sample is more than 24 hours or the storage time is more than 1 year, the DNA will be fragmented too much and the target band cannot be amplified.

3. The samples should be completely dehydrated before embedding to prevent the residual formalin from affecting the follow-up experiments.

4. When sampling embedded tissue, we should remove the excess paraffin and cut up the sample as much as possible, and the sampling amount should not exceed 30 mg, otherwise it is easy to cause insufficient lysis and affect the yield of nucleic acid.

5. When the extracted genomic DNA is used as a template for PCR amplification, it is suggested that the template gradient test should be done first and the best template concentration should be selected for amplification.

6. Please wear a lab coat and disposable gloves when operating.

For Research Use Only!

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