Fast RT-PCR kit for MeDIP / hMeDIP & robust post-bisulfite DNA library prep

news June 07 2021

 

Rapid, Sensitive & Specific Quantitative Real-Time PCR Analysis of MeDIP & hMeDIP

EpiQuik Quantitative PCR Fast Kit

For quantitative real time PCR analysis of ChIP, hmeDIP, meDIP, and meRIP DNA.

Product Overview

The EpiQuik™ Quantitative PCR Fast Kit allows for a fast, specific, sensitive, and reproducible quantitative real time analysis of DNA samples. The novel hot start DNA polymerase contained in this kit reduces the overall time required for quantitative PCR from approximately 2 hours to just 70 minutes. The kit also facilitates sensitivity and specificity of quantitative PCR by significantly increasing primer-DNA template annealing, while simultaneously reducing non-specific annealing. This results in considerable time savings and a more efficient real time PCR, particularly for ChIP, meDIP, hmeDIP, and meRIP samples.

An extremely fast procedure that can be finished within 70 minutes.
Abundant yields due to high amplification efficiency.
Highly accurate and specific in PCR, which reduces false-positive results.
Convenient master mix format allows for an easy reaction setup.
Can be used with any block-based real-time PCR instrument.
Simple, reliable, and consistent assay conditions.

Principle & Procedure

The EpiQuik™ Quantitative PCR Fast Kit provides a master mix format which contains a hot start DNA polymerase, dNTPs, a PCR enhancer, an optimized buffer, and an intercalating green dye. This master mix allows for a convenient and easy reaction setup. The unique hot start DNA polymerase is provided in an inactive state at ambient temperature and is reactivated by several minute long incubations at 95°C, which can easily be integrated into existing thermal cycling steps. The hot start DNA polymerase in combination with the optimized buffer ensures the quantitative PCR specificity and sensitivity. The green dye allows for DNA detection and analysis without using a sequence-specific probe.

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Prepare a DNA Library in Just 5 Hours After Successful Bisulfite Conversion

EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina)

For constructing Illumina-compatible libraries directly from bisulfite-treated DNA for Methyl-Seq.

Product Overview

The EpiNext™ Post-Bisulfite DNA Library Preparation Kit is a complete set of optimized reagents to prepare a DNA library — after successful bisulfite conversion — for various Illumina platform-based bisulfite sequencing (bisulfite-seq) assays, such as whole genome bisulfite sequencing (WGBS), oxidative bisulfite sequencing (oxBs-seq), reduced representation bisulfite sequencing (RRBS), and other bisulfite-based next generation sequencing applications. The optimized protocol and components of the kit allow both non-barcoded (singleplexed) and barcoded (multiplexed) DNA libraries to be quickly constructed using sub-nanogram input concentrations of DNA since the DNA is first bisulfite-converted and then used for library preparation. The kit has the following advantages and features:

  • Allows bisulfite-converted DNA to be used directly for ligation, thereby eliminating the possibility of breaking adapter-ligated fragments, which can often occur in currently used WGBS and RRBS methods.
  • Fast 5-hour procedure, from input starting material to library amplification.
    Gel-free size selection/purification saves time and prevents handling errors, as well as loss of valuable samples.
  • High sensitivty and efficiency — direct ligation of adapter to bisulfite-converted DNA fragments reduces loss of fragments and selection bias, which enables pre-bisulfite input DNA to be as low as 1 ng. The kit can be used for both non-barcoded (singleplexed) and barcoded (multiplexed) DNA library preparation.
  • Comprehensive set of components to accommodate each step of DNA library preparation — ligation, clean-up, size selection, and library amplification — for convenience, consistency, and reliability.
  • Ultra HiFi amplification enables achievement of reproducibly high yields of DNA libraries with minimal sequence bias and low error rates.

Principle & Procedure
With this kit, bisulfite-treated DNA (which is in single-stranded form), is converted to double-stranded DNA and directly used for ligation with BisDNA-specific adapters which are necessary for amplification and sequencing. The fragments are then size selected and purified with MQ beads, which allows for quick and precise size selections of DNA. Size-selected DNA fragments are then amplified with a high-fidelity PCR Mix, ensuring maximum yields from minimum amounts of starting material and providing a highly accurate amplification of library DNA with low error rates and minimal bias.

Starting Materials
Input starting material must be bisulfite-treated DNA generated from various input DNA amounts of 1 ng to 1 µg. For optimal preparation, the input DNA amount for the bisulfite conversion process should be 100 ng to 200 ng so that sufficient bisulfite-treated DNA can be yielded.

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