Paraffin-embedded Tissue DNA Extraction Kit

1. Components of the reagent kit

2. Storage

This reagent kit should be stored at room temperature (15-25℃) and in dry conditions, with a shelf life of 12 months. The DNA extraction purification columns can be stored for 1 year in a cool and dry environment. Proteinase K and RNase A contain preservatives, allowing transportation at room temperature, but for long-term storage, they should be kept at -20℃.

3. Instructions for Using the Reagent Kit

3.1 This reagent kit is intended for molecular biology research and should not be used for disease diagnosis or treatment.

3.2 Some components in the reagent kit contain irritants. Protective measures such as wearing protective clothing and goggles are recommended.

3.3 During the usage of this reagent kit, a high-speed centrifuge, water bath (metal bath), vortex mixer, anhydrous ethanol, sterile deionized water, and EP tubes need to be prepared by the user.

4. Introduction to the Reagent Kit

Paraffin-embedded tissue DNA purification kit provides a rapid and effective DNA purification solution for samples in paraffin sections. Tissues are collected after paraffin dissolution using a dedicated dewaxing solution, and sample DNA is obtained through subsequent extraction processes.

This kit can extract sample DNA from paraffin sections within 30 minutes, and the entire purification process does not require toxic reagents such as phenol-chloroform. The extracted DNA can be directly used for PCR, Southern blotting, and other applications.

5. Experimental Principles and Procedures

6. Extraction Process

Before Starting the Experiment:

A. Reagent Buffer FF:This buffer should be stored long-term in an environment between 2°C and 8°C.

B. Reagent Buffers IV and C may precipitate under low-temperature conditions. It is recommended to heat at 65°C for 5 minutes. After the precipitate dissolves, the solution can be used normally.

C. WashBuffer 1 should have a specified amount of anhydrous ethanol added before use, as indicated on the reagent bottle label. Check the label with a tick mark to indicate the addition of anhydrous ethanol.

D. Elution Buffer is a 0.1x TE solution with a minimal amount of EDTA. If EDTA affects subsequent experiments, it is advisable to replace the elution buffer with sterile deionized water.

1. Sample Processing:

Select 5-10 paraffin sections (1mm-3mm thickness), use scissors to remove excess wax, and cut the embedded tissue sample into small pieces. Place them in a 1.5ml centrifuge tube, add 800μl Reagent Buffer FF, incubate at 75°C for 5 minutes until all paraffin is dissolved. Centrifuge at 12000rpm for 5 minutes, discard the supernatant.

2. Add 400μl Reagent Buffer IV, 20μl Proteinase K (10 mg/ml), and 10μl RNaseA (10 mg/ml) to the precipitate from the previous step. Digest at 65°C, stirring every 6-7 times until digestion is complete.
3. Add an equal volume of Reagent Buffer Cand an equal volume of anhydrous ethanol, mix thoroughly by pipetting.

(For example: If you add 400μl Reagent Buffer IV, approximately add 430μl Reagent Buffer C and 430μl anhydrous ethanol. Some precipitation may occur after adding Reagent Buffer C, but it does not affect subsequent experiments.)

4. Add the obtained liquid to the DNA extraction purification column (or cartridge) (approximately 650-700μl each time), let it stand at room temperature for 2 minutes, centrifuge at over 8,000rpm for 1 minute. Discard the collected waste and reinsert the collection tube into the purification column for the next step.
5. Repeat step 4, adding the remaining liquid to the DNA extraction purification column (or cartridge), centrifuge at over 8,000rpm for 1 minute, discard the waste and the collection tube.
6. Place the DNA extraction purification column (or cartridge) into the collection tube, add 300μl WashBuffer 1, centrifuge at over 8,000rpm for 1 minute. Discard the waste and reinsert the purification column into the tube for the next step.

(Note: Confirm that anhydrous ethanol has been added to Wash Buffer 1.)

7. Add 500μl WashBuffer 1 to the DNA extraction purification column (or cartridge), centrifuge at 14,000rpm (20000×g) for 2 minutes. Extend centrifugation time if needed for a dryer membrane.
8. Place the DNA extraction purification column (or cartridge) in a new centrifuge tube, open the lid, incubate at 65°C for 2 minutes. Extend this step if necessary to evaporate ethanol to prevent residual ethanol from affecting downstream experiments.
9. Suspend the addition of 50-100μl Elution Buffer to the membrane, centrifuge at 12,000rpm for 2 minutes.

(Note: 1. Using 50μl Elution Buffer for DNA elution can increase DNA concentration but decreases total DNA yield. 2. The eluted DNA can be reapplied to the DNA extraction purification column, centrifuged at 12000rpm for 2 minutes again to increase DNA yield.)