MethylGold DNA Bisulfite Conversion Kit

Epigenetics is a branch of genetics that studies the heritable changes in gene expression and regulation without changing the nucleotide sequence of genes. Among them, DNA methylation is the earliest discovered and one of the most deeply studied epigenetic regulation mechanisms.

Bisulfite conversion reaction is a common technique to study the methylation pattern of genomic DNA. The product combines bisulfite conversion reaction with new silica gel membrane technology to convert unmethylated cytosine to uracil, while methylated cytosine remains unchanged, and the conversion efficiency can reach more than 99%. The transformed DNA can be used in downstream analytical experiments, including PCR amplification, restriction endonuclease digestion, sequencing, microarray, etc.

Buffer BP is shipped with wet ice and stored at -20°C. Other reagents are shipped and stored at room temperature; valid for 12 months.

1. Add 280 μL Buffer BM, 350 μL BP and 560 μL Nuclease-free Water to CT Conversion Reagent before use, then vortex to dissolve and stored at -20°C.

2. Please add 12 mL isopropanol to Buffer GB before use, mix well and use.

3. Please add 24 mL anhydrous ethanol to Buffer DB before use, add 80 mL anhydrous alcohol to Buffer PW, mix well and use.

1. Column balance: Place the DNA Spin Column in the Collection Tube, add 500 μL Buffer BL. Centrifuge at 12,000 rpm for 1 min, discard the filtrate in Collection Tube, and put the DNA Spin Columns back into the Collection Tube (treated DNA Spin Column are best used on the same day).

2. Prepare the bisulfite conversion system in a 200 μL centrifuge tube: Transfer 20 μL genomic DNA into a centrifugal tube (the total amount is 500~2000 ng, if less than 20 μL can be supplemented with Nuclease-free Water), add 130 μL CT Conversion Reagent, and gently mix with a pipette.

3. Transfer the mixture to the thermal cycle instrument, incubate at 98°C for 10 min, 64°C for 2.5 h and store at 4°C (optional).

4. After the end of the reaction, briefly centrifuge, collect the wall solution at the bottom of the pipe, transfer the reaction solution to the 1.5 mL centrifuge tube.

5. Add 600 μL Buffer GB (confirm whether isopropanol is added) and mix well with a pipette.

6. Transfer the solution obtained in the previous step to DNA Spin Column, centrifuge at 12,000 rpm for 1 min, discard the filtrate in Collection Tube, and put the DNA Spin Columns into the Collection Tube.

7. Add 600 μL Buffer PW to DNA Spin Column (confirm whether or not to add anhydrous ethanol). Centrifuge at 12,000 rpm for 1 min, discard the filtrate in Collection Tube, and put the DNA Spin Columns into the Collection Tube.

8. Add 600 μL Buffer DB to DNA Spin Column (confirm whether or not to add anhydrous ethanol). Place at room temperature for 15 min, centrifuge at 12,000 rpm for 1 min, discard the filtrate in Collection Tube, and put the DNA Spin Columns into the Collection Tube.

9. 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, discard the filtrate in Collection Tube, and put the DNA Spin Columns into the Collection Tube.

10. Repeat step 9.

11. Centrifuge at 12,000 rpm for 2 min, put DNA Spin Column into a new 1.5 mL centrifuge tube.

12. Open the centrifuge tube lid, leave it at room temperature for 5~10 min to completely volatilize ethanol.

13. Add 20 μL Nuclease-free Water to the DNA Spin Column, stand at room temperature for 2 min.

14. Centrifuge at 12,000 rpm for 2 min, the transformed DNA can be obtained.

1. Please read the Product Manual carefully before use.

2. The addition of Buffer BL in the column balance step can improve the adsorption capacity of the adsorption column, improve the uniformity and stability of the adsorption column, and eliminate the influence of high temperature, humidity or other adverse environmental factors on the adsorption column.

3. The processing time of Buffer DB should not be too long, it is easy to cause excessive fragmentation of DNA and affect the follow-up experiments.

4. If the recycled DNA is used on the same day, please keep it at 2~8°C. If you can't use it in time, please keep it at -20°C.

5. In the subsequent PCR amplification reaction, it is suggested that 2~3 μL products should be added as templates in the 20 μL system.

6. For your safety and health, please wear a lab coat and disposable gloves.

For Research Use Only!