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BisulFlash DNA Modification Kit

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For lightning fast bisulfite treatment of DNA for methylation-specific PCR analysis

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Suggested Workflow
DNA Isolation
 
 
DNA Bisulfite Conversion
 
 
PCR Analysis
 
Complete Cytosine Conversion: 200 ng of genomic DNA isolated from 3 cancer cell lines was treated with the BisulFlash™ DNA Modification Kit. Next, the unconverted and converted DNA in each treated sample were determined using unconverted DNA-specific and converted DNA-specific primers (β-actin, 110 bps), respectively. A: real time PCR; B: end-point PCR. The BisulFlash™ kit treated DNA was completely converted, and no unconverted DNA in the treated samples was determined after 45 cycles.
Effective DNA Protection: Fully methylated human genomic DNA at various amounts (0.2 ng-200 ng) were converted using the BisulFlash™ DNA Modification Kit. 1
High accuracy of DNA conversion is achieved by the BisulFlash™ DNA Modification Kit. 50 ng of genomic DNA methylated in all CpG sites by DNA methylases was treated with the BisulFlash™ DNA Modification Kit. Converted DNA was amplified by real time qPCR using primers for multiple promoters containing numerous CpG sites and then directly sequenced.
Schematic procedure of the BisulFlash™ DNA Modification Kit to obtain converted DNA.
Input Type: DNA
Research Area: DNA Methylation
Target Application: Sample Modification
Vessel Format: Columns/Tubes
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-1026-05050 reactions $122.00 
Availability: Usually Ships in 1 Day or Same day delivery Same Day NY Delivery 
Product Overview

The BisulFlash™ DNA Modification Kit is a complete set of optimized buffers and reagents to perform DNA modification using an expedited DNA bisulfite conversion technology developed by Epigentek, geared specifically for methylation-specific PCR (MSP) as well as post-bisulfite based NGS applications. Through a proprietary composition which allows DNA denaturation and bisulfite conversion to be processed at the same time, the complete procedure is reduced to only 30 minutes. Furthermore, it prevents more than 90% of DNA loss, completely converting unmethylated cytosine into uracil.

Perfecting Bisulfite Conversion
Traditional methods involve a separate denaturation step followed by a subsequent sodium bisulfite DNA conversion step -- but with the BisulFlash™ method, DNA denaturation status is concurrently sustained throughout the entire DNA bisulfite conversion process. This breakthrough approach enables the DNA conversion process to be significantly faster with higher conversion efficiency and accuracy. We continue to innovate with the development of the new BisulFlash™ kit by identifying four critical components of bisulfite conversion:
  • Speed: Reduce the entire procedure to as short as 30 minutes without any reagent setup time.
  • Efficiency: Completely convert unmethylated cytosine into uracil -- modified DNA > 99.9%.
  • DNA Protection: Protect against DNA degradation of which more than 90% of DNA loss can be prevented, allowing for greater recovery.
  • Sensitivity: Start with the lowest amount of input DNA for modification -- only 0.2 ng or just 50 cells.

The convenient ready-to-use, DNA conversion mix solution and single temperature incubation along with the features mentioned above allow for true perfection in bisulfite conversion. The BisulFlash™ DNA Modification Kit is suitable for MS-PCR/MSP and real time MSP.

Bisulfite Sequencing Option
The BisulFlash™ kit includes a special step made specifically for better sequencing results and is 100% compatible with all Illumina® platforms. For ideal NGS applications, we recommend combining this technique with our EpiNext Post-Bisulfite Library Preparation Kit.

Fig. 1. High accuracy of DNA conversion is achieved by the BisulFlash™ DNA Modification Kit. 50 ng of genomic DNA methylated in all CpG sites by DNA methylases was treated with the BisulFlash™ DNA Modification Kit. Converted DNA was amplified by real time qPCR using primers for multiple promoters containing numerous CpG sites and then directly sequenced.

Comparative Overview of Commercial Kits
Data was obtained through actual use of the kit, customer feedback, or information provided by the supplier's datasheet or website.

  BisulFlash Supplier 1 Supplier 2 Supplier 3
Processing Time 30 min >3 hours >6 hours 12-16 hours
Correct Conversion 99.9% 99.5% 99.4% 98%
Error Conversion <0.1% >0.3% N/A >1.5%
Correct/Error Conversion Ratio ≅ 1000 ≅ 330 N/A ≅ 65
DNA Degradation Very low Medium Low High
Minimum Starting DNA 0.2 ng 0.5 ng 1 ng 1 ng
Convenience Very high High Medium Low
 
Product Details

Principle & Procedure
As a next generation bisulfite conversion tool, the BisulFlash™ DNA Modification Kit contains all reagents required for an ultra-fast bisulfite conversion on a DNA sample. With the ready-to-use conversion mix solution, DNA denaturation status is sustained throughout the entire bisulfite DNA conversion process. This proprietary solution allows the bisulfite reagents to rapidly convert all cytosine to uracil with negligible methylcytosine conversion. The unique DNA protection reagents contained in the mix can prevent the chemical and thermophilic degradation of DNA in the bisulfite treatment. The non-toxic DNA capture solution enables DNA to tightly bind to the column filter, thus DNA cleaning can be carried out on the column to effectively remove residual bisulfite and salts.

Rapid Results
Only 30 minutes are required for the entire BisulFlash™ procedure -- from converting your sample DNA to pure bisulfite modified DNA. That is significantly faster than any currently used kits (2-8 h) or homebrew methods (>16 h). The BisulFlash™ kit provides everything required for a successful bisulfite conversion and DNA cleanup in the shortest time with the fewest steps possible.

Perfect DNA Conversion
Each reaction with the BisulFlash™ kit can use 0.2 ng – 1 µg of DNA. For optimal conversion, the DNA amount is 50-200 ng. The novel procedure and proprietary ready-to-use DNA conversion mix solution allow DNA denaturation and bisulfite conversion to occur at the same time and enable all cytosines to be converted to uracil (>99.9%), while 5-methylcytosine remains the same. The highly efficient cytosine conversion is proven by the low CT values obtained when amplifying converted DNA using real-time PCR (Fig. 2). This perfect conversion rate is superior to other bisulfite kits available on the market and provides repeatable and dependable downstream analysis.

Powerful DNA Protection
DNA protection reagents are added into the DNA conversion mix solution to prevent DNA from chemical and thermophilic degradation in the bisulfite treatment and provide effective DNA denaturation, resulting in single-stranded DNA necessary for complete cytosine conversion. The prevention of DNA degradation enables subsequent amplification and analysis of large PCR fragments (Fig. 3). The efficient integrated DNA cleanup and unique elution buffer allow for long-term storage (> 6 months) and multiple freezing/thawing of the converted DNA without affecting the DNA quality.


Fig. 3. Complete Cytosine Conversion: 200 ng of genomic DNA isolated from 3 cancer cell lines was treated with the BisulFlash™ DNA Modification Kit. Next, the unconverted and converted DNA in each treated sample were determined using unconverted DNA-specific and converted DNA-specific primers (β-actin, 110 bps), respectively. A: real time PCR; B: end-point PCR. The BisulFlash™ kit treated DNA was completely converted, and no unconverted DNA in the treated samples was determined after 45 cycles.

Fig. 4. Effective DNA Protection: Fully methylated human genomic DNA at various amounts (0.2 ng-200 ng) were converted using the BisulFlash™ DNA Modification Kit. 1 µl of 20 µl eluate was used for real time qPCR and a pair of primers was used to amplify converted DNA. As little as 0.2 ng DNA is sufficient for bisulfite conversion using the BisulFlash™ DNA Modification Kit. A: real time PCR; B: starting DNA amount-CT value curve.
Product Components

BF1 (Conversion Mix Solution)
BF2 (Capture Solution)
BF3 (Desulphonation Solution)
BF4 (Elution Solution)
BF5 (Conversion Enhancer)
BF6 (Denaturation Enhancer)
F-Spin Columns
F-Collection Tubes
User Guide

User Guide & MSDS

[User Guide]*
*Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

Product Citations

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Customer Reviews

Rating By c********@niehs.nih.gov Verified Purchase Reviewed on: Tuesday 12 October, 2010
Application Description
Pyrosequencing from blood samples

Other Thoughts
Kit was really good for higher starting amounts, not as good as others for lower amounts. If starting quantity not limiting, good kit to use.
Rating By m*****@oakland.edu Verified Purchase Reviewed on: Tuesday 12 October, 2010
Application Description
We have used your kit to make converted DNA from immature and mature neural retina to do Methylation specific PCR of the Rhodopsin gene. (off in immature, and on in mature) We can successfully confirm hypomethylation changes using DNA we processed with your kit. I have thus mentioned your kit specifically in an NIH grant application I just submitted last Monday. (R01).

Procedural Details
We did not that while a range of DNA can be processed with your kit, in reality the upper end of DNA range is best and often necessary. So processing 1 ug of DNA per treatment/column, is best to ensure that qPCR detection works well. Amounts lower than 500 ng would often fail for qPCR unless we did a precip and concentration. 1 ug input into the treatment ensures no need to reconcentrate treated DNA and successful qPCR with 2 ul per qPCR reaction. We used Amplitaq gold, gold buffer, and sybr green we add ourselves in an MX3000 qPCR unit.

Other Thoughts
Methylation primers and un-methylated primer reactions each support each other. (methyl decreases as unmethy increases). Differences between ON and OFF gene state were well in excess of 10 fold, making the differences quite clear to us. We also know what to expect from previous qChIP analysis of 5-methylcytosine.
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