Superfast DNA Methylation Bisulfite Kit HYD115
Superfast DNA Methylation Bisulfite Kit is a next-generation enzymatic library preparation kit designed for use with Illumina and MGI high-throughput sequencing platforms. Compared to traditional library preparation methods, this kit utilizes high-quality fragmentation enzymes, eliminating the need for cumbersome sonication processes. The fragmentation and end-repair modules are combined into a single step, significantly reducing both the time and cost of library preparation. This kit is suitable for a wide range of sample types, including plant and animal genomes, microbial genomes, and more, with a sample input range of 1 ng to 1 μg. The enzymatic fragmentation ensures uniform fragment sizes across different species, with minimal inter-species variation. Additionally, this kit can be paired with Illumina or MGI adapters and primers for sequencing on Illumina or MGI high-throughput platforms.
Reagent Composition
|
Components |
50T |
|
Transformation solution |
1.5 mL×7 |
|
Wash solution* |
5.5 mL×1 |
|
Desulfurization solution |
11 mL×1 |
|
Elution solution |
1.5 mL×1 |
|
Purification column |
10 ×5 |
|
Collection tube |
10 ×5 |
* Add 22 mL of anhydrous ethanol to the washing solution;After adding anhydrous ethanol, invert and mix well and set aside. At this time, the bottle cap should be tightened to prevent ethanol from volatilizing and affecting the use of the reagent.
Storage Conditions
The conversion solution should be stored below 25℃ and away from light. Other components should be stored at room temperature. The shelf life is 1 year.
Notes
1. To ensure the smooth progress of downstream experiments, the total amount of DNA input should be accurately quantified during the conversion step, and the A260 / A280 ratio should be between 1.7-1.9;
2. The DNA input range is 100 pg-2 μg, and the optimal input is 100 ng-1 μg. Too low is not conducive to downstream detection, and too high may lead to reduced recovery rate/conversion efficiency.
3. The Transformation solution/Desulfurization solution/Wash solution contains volatile components. After use, the bottle cap should be tightened in time and stored at room temperature. The storage temperature of the conversion solution should not exceed 25℃ as much as possible, and one tube of conversion solution should be used up at one time as much as possible.
4. After conversion, the sample should be subjected to downstream experiments in time. If it is stored for a short time, it can be placed at -20℃, and it can be placed at -80℃ for long-term storage.
5. For your safety and health, please wear a lab coat and disposable gloves when operating.
6. This product is for scientific research purposes only!
Instructions
1.Preparation of reagents and consumables: 1.5 mL sterile centrifuge tubes, sterile pipette tips, nuclease-free water, anhydrous ethanol, PCR tubes;
2.Bisulfite conversion:
2.1 Prepare the corresponding sterile PCR tubes according to the number of samples required for the experiment, and prepare the reaction system according to the requirements in the following table:
2.2 Conversion system
|
Components |
Volume |
|
Input DNA |
100 pg-2 μg |
|
Transformation solution |
5.5 mL×1 |
|
ddH2O |
Up to 200μL |
Use a pipette to mix the above system by pipetting or vortexing briefly for 5 seconds, centrifuge briefly, and centrifuge the reaction solution to the bottom of the PCR tube.
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 aliquoted and transferred to a new sterile PCR tube after pipetting and mixing in step 2) to run the transformation program. After the transformation is completed, the reaction solutions in the two PCR tubes are combined into the same purification column for purification.
2. If the sample volume is between 20-40 μL, reduce the volume of the transformation solution and keep the total volume at 200 μL.
3. If the sample volume is 50 μL, add 150 μL of the transformation solution, keep the total volume at 200 μL, and extend the transformation time to 6-10 min.
2.3 CT conversion program settings
|
Temperature |
Time |
|
98 ℃ |
5 min* |
|
4 ℃ |
∞ |
3. Purification of transformation products
3.1 Prepare a purification column kit for the corresponding number of samples, transfer 200 μL of the reaction solution after transformation to the purification column, centrifuge at 13000 g for 30-60 s, discard the filtrate, and put the purification column back in the collection tube;
3.2 Add 100 μL of wash solution to the purification column (make sure that anhydrous ethanol has been added), centrifuge at 13000 g for 30-60 s, discard the filtrate, and put the purification column back in the collection tube;
3.3 Add 200 μL of desulfurization solution to the purification column, let it stand at room temperature for 20 min, centrifuge at 13000 g for 30-60 s after the reaction, discard the filtrate, and put the purification column back in the collection tube;
3.4 Add 200 μL of wash solution to the purification column, centrifuge at 13000 g for 30-60 s, discard the filtrate, and put the purification column back in the collection tube;
3.5 Repeat steps 4) once;
3.6 Transfer the purification column to the prepared 1.5 mL centrifuge tube, open the lid and dry it, add 10-30 μL of elution solution to the center of the filter membrane, let it stand at room temperature for 1 minute, centrifuge at 13000 g for 1 minute, and collect the eluent;
3.7 Store the eluted sample at -20℃ temporarily. If it is stored for a long time, please store the sample at -80℃, and avoid unnecessary repeated freezing and thawing. Note: The purified conversion product can be directly used in subsequent PCR reactions or sequencing processes.