Hieff Superfast DNA Methylation Bisulfite Kit _ 12225ES
Roll over image to zoom in Click on image to zoom
Hieff Superfast DNA Methylation Bisulfite Kit _ 12225ES

Hieff Superfast DNA Methylation Bisulfite Kit _ 12225ES

Save $45.00
SKU: 12225ES10
Size: 10T
Price:
$105.00 $150.00

Shipping calculated at checkout

Stock:
In stock
Bulk Quote Inquiry

Description

The Hieff Superfast DNA Methylation Bisulfite Kit (Column-based) rapidly converts unmethylated cytosines in DNA samples to uracil, while leaving methylated cytosines unchanged. During high-temperature bisulfite treatment, double-stranded DNA denatures into single strands. In the presence of HSO3-, cytosine residues undergo deamination and are converted into uracil, with methylated cytosines remaining unaltered. In subsequent PCR amplification, uracil is replaced by thymine (T). The conversion process takes only 5 minutes, accommodating DNA input ranging from 100 pg to 2 μg, and achieves a conversion efficiency of ≥99% for unmethylated cytosines. The converted DNA is suitable for downstream applications such as PCR amplification and NGS sequencing.

Feature

Low Input: Suitable for converting samples ranging from 100 pg to 2 μg

Short conversion time: approximately 5 minutes.

Minimal Sample Damage: Maintaining good sample integrity post-conversion. 

High Conversion Efficiency: Conversion rate ≥99%,  high conversion rates in high-GC regions with a low false positive rate. 

Suitable for rare samples, such as single-cell DNA methylation conversion.

Capable of methylation conversion of RNA samples.

 

Product Components 

No.

Component name

12225ES10

12225ES50

12225-A

Conversion Reagent

2 mL×1

3.3 mL×3

12225-B

Wash Buffer

1.1 mL×1

5.5 mL×1

12225-C

Desulphonation Buffer

2.2 mL×1

11 mL×1

12225-D

Elution Buffer

500 μL×1

1.5 mL×1

12225-E

DNA Column

10

50

12225-F

Collection Tube

10

50
Note: 

For 10 tests per box: Add 4.4 mL of anhydrous ethanol to the washing solution.

For 50 tests per box: Add 22 mL of anhydrous ethanol to the washing solution.

After adding the anhydrous ethanol, invert and mix well, then store for future use. Ensure the bottle cap is tightly closed to prevent ethanol evaporation, which could affect reagent performance.



Figure

Figure 1. The DNA samples used for testing included 300 ng of unmethylated Lambda DNA and 800 ng of human genomic DNA. Post-conversion, Qubit ssDNA quantification was employed with an elution volume of 30 μL.

Figure 2. The converted products from 800ng of human genomic DNA were then subjected to methylation-specific primer probe PCR targeting a 286 bp fragment size. Gel electrophoresis was used to analyze the amplification products . 
Figure 3. A direct comparison between the 12225 conversion kit and competitor kits Q and Z. The samples used for conversion comprised 200 ng and 2 μg of FFPE DNA, with an added 1% λDNA. Following conversion, the samples underwent methylation-specific single-stranded DNA library preparation.

Figure 4. Bisulfite conversion on human genomic DNA samples were performed using both the 12225 and competitor Q conversion kits. Subsequently, methylation-specific single-stranded DNA library preparation kits were utilized to generate libraries. The library yield and quality control data are presented above. The results demonstrate that when sequencing the pooled libraries from both the 12225 and competitor Q kits, the 12225 kit yielded higher data output, achieved a higher on-target ratio (%), and exhibited lower duplication rates (%).

Shipping and Storage

Store 12225-A conversion solution at room temperature, protected from light. Store other components at room temperature. The shelf life is 12 months.

Note:


1. To ensure the success of downstream experiments, accurately quantify the total amount of input DNA during the conversion step. It is recommended to use Qubit 3.0/4.0 for DNA quantification, with an A260/A280 ratio between 1.7 and 1.9. The input DNA range should be between 100 pg and 2 μg, with an optimal range of 100 ng to 1 μg. Insufficient DNA input can hinder downstream detection, while excessive input may reduce recovery and conversion efficiency.

2. The conversion solution, desulfonation solution, and washing solution contain volatile components. After use, promptly tighten the caps and store at room temperature.

3. For post-conversion samples, proceed with downstream experiments promptly. For short-term storage, keep at -20°C, and for long-term storage, keep at -80°C.

4. For your safety and health, wear a lab coat and disposable gloves during operation.

5. This product is for research use only!


Instructions

Preparation of reagents and consumables: 1.5 mL sterile centrifuge tube, enzyme-free water, absolute ethanol, PCR tube;

l Bisulfite conversion:

1) Prepare the corresponding sterile PCR tube according to the number of samples to be tested, and prepare the reaction system according to the following table:

2) Transformation system

Component

Volume

DNA

100 pg-2 μg (to 20 μL)

Conversion Buffer

180 μL

Total volume

200 μL

Use a pipette to blow and mix the above system or vortex and mix it for 5 s, then centrifuge it for a short time, and centrifuge the reaction solution to the bottom of the PCR tube.

v Note: 1. At this time, the total volume of the reaction solution in the PCR tube is 200 μL. In order to make the transformation more complete, the reaction solution should be divided into equal parts and transferred to a new sterile PCR tube after blowing and mixing in step 2), and the transformation procedure should be run. After the conversion, the reaction solutions in the two PCR tubes were combined into the same purification column for purification.

2. If the sample volume is between 20 and 40 μL, reduce the volume of the transformation solution to maintain a total volume of 200 μL.

3. If the sample volume is 50 μL, 150 μL of the transformation solution is added, the total volume is maintained at 200 μL, and the transformation time is extended to 6-10 min.

3) Setting of CT conversion program

Temperature

Time

98 ℃

5 min

4 ℃

∞

The PCR tube is placed on a PCR instrument with a preset program to carry out the reaction.

l Purification

1) Transfer 200 μL of the conversion solution to the Purification columns, centrifuge for 30-60 s 13000 g, discard the filtrate, and put the purification column into the Collection tubes again;

2) Add 100 μL of Wash Buffer into Purification Columns (confirm that absolute ethanol has been added), centrifuge for 30-60 s 13000 g, discard the filtrate, and put the purification column into the Collection tubes again;

3) Add 200 μL of Desulphonation Buffer into the Purification Columns , and allow the reaction to stand at room temperature for 20 min. After the reaction, centrifuge for 30-60 s 13000 g, discard the filtrate, and place the Purification Columns in the Collection tubes again;

4) Add 200 μL of the Wash Buffer to the Purification Columns, centrifuge for 30-60 s 13000 g discard the filtrate, and place the Purification columns in the Collection tubes again;

5) Repeat step 4) once;

6) Transfer the Purification Columns to the prepared 1.5 mL centrifuge tube, add 10-30 μL of Elution Buffer to the center of the filter membrane after opening the cover and drying, and collect the DNA after standing for 1 min at room temperature and centrifuging for 1 min at 13000 g;

7) Store the DNA temporarily at -20 ℃. For long-term storage, please store the DNA at -80 ℃ and avoid unnecessary repeated freezing and thawing.

Note: The purified transformation product can be directly used in the subsequent PCR reaction or sequencing process.

 

Manual

Yeasen Inquiry

You may also like