MagBind MethylGold DNA Bisulfite Conversion Kit

 

Product Name	Cat.No.	Spec.
MagBind MethylGold DNA Bisulfite Conversion Kit	G3639-50T	50 T

 

 

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. In many animals and plants, DNA methylation is based on S-AdenosylMethionine as methyl donor, under the catalysis of methyltransferase, the fifth carbon position of cytosine ring covalently binds to a methyl group.

DNA methylation is a naturally occurring event in prokaryotes and eukaryotes. In prokaryotes, DNA methylation protects host DNA from being digested by restriction endonucleases, which can eliminate exogenous DNA. In higher eukaryotes, DNA methylation plays an important role in the regulation of gene expression.

The product uses bisulfite to treat methylated DNA and converts unmethylated cytosine into uracil, while methylated cytosine remains unchanged, the whole process can be completed within 4 hours, and the conversion efficiency can reach more than 99%. At the same time, the kit adopts the method of desulphurization and recovery of DNA on magnetic beads, which can effectively improve the recovery amount of transformed DNA, and the whole operation flow is very simple. The transformed DNA can be used for PCR amplification and downstream analysis, including restriction endonuclease digestion, sequencing, microarray and so on.

 

 

Shipped and stored at room temperature; valid for 12 months.

 

 

Component Number	Component	G3639-50T
G3639-1	CT Conversion Reagent	10 times / tube×5
G3639-2	Buffer BM	1.5 mL
G3639-3	Buffer GB	10 mL
G3639-4	Buffer DB	3.6 mL (8.4 mL of anhydrous ethanol added before use)
G3639-5	Buffer PW	24 mL (56 mL of anhydrous ethanol added before use)
G3639-6	SweMag Beads	1 mL
G3639-7	Nuclease-free Water	10 mL
Manual		One copy

 

 

1. Add 280 μL Buffer BM and 910 μL Nuclease-free Water to CT Conversion Reagent before use, then vortex to dissolve and stored at -20°C.

2. Please add 8.4 mL anhydrous ethanol to Buffer DB before use, add 56 mL anhydrous alcohol to Buffer PW, mix well and use.

3. Magnetic stands and isopropanol are required but not supplied in this kit.

 

 

1. Add 20 μL genomic DNA (the total amount is 500 ng-2000 ng, and if the volume is less than 20 μL, it can be supplemented by Nuclease-free Water) to PCR tube. Add 130 μL CT Conversion Reagent and gently mix with a pipette.

2. Transfer the mixture of the first step to the PCR instrument and set it at 98°C for 10 min and 64°C for 2.5h. After operation, the product is stored at 4°C.

3. Add 150 μL Buffer GB and 130 μL isopropanol to the above products, use a pipette to mix well, then add 15 μL SweMag Beads (Vortex to evenly dispersed before use), and use a pipette to blow until the beads are uniformly dispersed.

4. Stand at room temperature for 10 min. During this period, mix evenly for 3~4 times with a pipette to keep the magnetic beads dispersed uniformly.

5. Move the centrifuge tube to the magnetic stand and let stand for 30 s, so that the magnetic beads are adsorbed to the wall of the tube. When it is clear, discard the supernatant (in order to avoid affecting the extraction efficiency, do not suck the magnetic beads out).

6. Remove the centrifuge tube from the magnetic stand, add 400 μL Buffer PW, blow with a pipette until the magnetic beads are dispersed evenly. Then move the centrifuge tube to the magnetic stand and let stand for 30 s, so that the magnetic beads are adsorbed to the wall of the tube. When it is clear, discard the supernatant (in order to avoid affecting the extraction efficiency, do not suck the magnetic beads out).

7. Remove the centrifuge tube from the magnetic stand, add 200 μL Buffer DB, blow it with a pipette until the magnetic beads are dispersed evenly, stand at room temperature for 15 min, mixing every 3~5 min during the period (Buffer DB processing time should not be too long, so as to avoid excessive fragmentation of genomic DNA). Move the centrifuge tube to the magnetic stand and stand for 30 seconds, so that the magnetic beads are adsorbed to the wall of the tube, and after it is clear, discard the supernatant (in order to avoid affecting the extraction efficiency, do not suck the magnetic beads out).

8. Remove the centrifuge tube from the magnetic stand, add 400 μL Buffer PW, blow it with a pipette until the magnetic beads are dispersed evenly. Then move the centrifuge tube to the magnetic stand and let stand for 30 s, so that the magnetic beads are adsorbed to the wall of the tube. When it is clear, discard the supernatant (in order to avoid affecting the extraction efficiency, do not suck the magnetic beads out).

9. Repeat step 8.

10. Open the centrifuge tube lid, leave it at room temperature for 5~10 min or at 65°C for 3~5 min to make the ethanol evaporate completely (do not make the magnetic beads too dry, so as not to affect the yield of nucleic acid).

11. Remove the centrifuge tube from the magnetic stand, add 30~50 μL Nuclease-free Water to the centrifuge tube, blow until the magnetic beads are dispersed evenly with a pipette, stand at room temperature for 5 min.

12. Move the centrifugal tube to the magnetic stand until the magnetic beads are completely adsorbed, and absorb the supernatant into a new centrifuge tube to obtain the converted DNA solution.

 

 

1. Please read the Product Manual carefully before use.

2. Magnetic beads are precipitate easily and should be shaken or vortex thoroughly before use.

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. Before elution, ethanol should be completely volatilized to avoid the influence of residual ethanol on downstream experiments.

5. Please do not dry the magnetic beads for a long time, as this may cause irreversible bead aggregation.

6. The recovered DNA should be stored at -20°C to avoid repeated freezing and thawing.

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

 

For Research Use Only!