dna methylation brochure 8 12 15 copy - caltag medsystems...the methylflash™ methylated dna (5-mc)...

DNA Methylation Complete Solutions for

DNA Isolation DNA Fragmentation

Methylated DNA Quantifi cationBisulfi te Conversion

Methylated DNA Immunoprecipitation

PCR & NGS Analysis

Complete Solutions for Epigenetics

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Methylated DNA Quantifi cationDNA Isolation

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DNA IsolationBisulfi te Conversion

PCR & NGS Analysis

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DNA IsolationDNA Fragmentation

Methylated DNA Immunoprecipitation

PCR & NGS Analysis

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Workfl ow of Contents

We o� er the most comprehensive selection of epigenetic research products to cover every step of your experiment work� ow, from upstream to downstream. Learn more at: www.epigentek.com/dna-methylation

Methylated DNA Quantifi cation Kits

Product NameMethylFlash

Methylated DNA Quantifi cation

MethylFlash Hydroxymethylated DNA

Quantifi cation

MethylFlash Urine 5-Methylcytosine (5-mC) Quantifi cation

MethylFlash 5-Formylcytosine (5-

fC) DNA Quantifi cation

Cat. # P-1034 P-1036 P-1039 P-1041

Detects 5-methylcytosine 5-hydroxymethylcytosine 5-methylcytosine 5-formylcytosine

Format strip-well microplate strip-well microplate strip-well microplate strip-well microplate

Starting Material DNA DNA Urine DNA

Input Range (Optimal Input) 50-200 ng (100 ng) 50-200 ng (100 ng) 0.5-5.0 µl (2 µl) 100-300 ng (300 ng)

Detection Threshold 200 pg of meDNA 40 pg of hMeDNA 30 pg of meDNA 1 pg of fC DNA

Specifi city >1000:1 meDNA:unMeDNA

>1000:1 hMeDNA:unMeDNA

>1000:1 meDNA:unMeDNA

>1000:1 fC DNA:unMeDNA

Time 4 hours 4 hours 4 hours 4 hours

3

MethylFlash Methylated DNA Quantifi cation KitCat. # P-1034

The MethylFlash™ Methylated DNA (5-mC) Quantifi cation Kit (Colorimetric)* is a complete set of optimized buffers and reagents to quantify global DNA methylation in DNA samples from any spe-cies by specifi cally measuring levels of 5-methylcytosine (5-mC) in an ELISA-like format, all in under 4 hours.

• Easy-to-follow steps - fi nish within 4 hours• Extremely low background signals• Recognizes as low as 0.2 ng of methylated DNA• No cross-reactivity to unmethylated or hydroxymethylated

cytosine• Flexible strip microplate format for manual or high throughput

Demonstration of high sen-sitivity and specifi city of meth-ylated DNA (5-mC) detection achieved by the MethylFlash™ kit. Synthetic unmethylated DNA (contains 50% of cyto-sine) and methylated DNA (contains 50% of 5-methyl-cytosine) were added into the assay wells at different concentrations and then mea-sured with the MethylFlash™ Methylated DNA Quantifi cation Kit (Cat. # P-1034).

MethylFlash Hydroxymethylated DNA Quantifi cation KitCat. # P-1036

The MethylFlash™ Hydroxymethylated DNA (5-hmC) Quantifi ca-tion Kit (Colorimetric)* is a complete set of optimized buffers and reagents to quantify global DNA hydroxymethylation in DNA samples by specifi cally measuring levels of 5-hydroxymethylcytosine (5-hmC) in an ELISA-like format, all in under 4 hours.

• Easy-to-follow steps - fi nish in less than 4 hours• Extremely low background signals• Recognizes as low as 0.04 ng of hydroxymethylated DNA• No cross-reactivity to unmethylated or methylated cytosine• Flexible strip microplate format for manual or high throughput

Demonstration of high sen-sitivity of 5-hydroxymethylcyto-sine detection achieved by the MethylFlash™ kit. Synthetic hydroxymethylated DNA was added into the assay wells at different concentrations and then measured with the Meth-ylFlash™ Hydroxymethylated DNA Quantifi cation Kit (Cat. # P-1036).

MethylFlash Urine 5-Methylcytosine Quantifi cation KitCat. # P-1039

MethylFlash 5-Formylcytosine DNA Quantifi cation KitCat. # P-1041

The MethylFlash™ Urine 5-Methylcytosine (5-mC) Quantifi ca-tion Kit (Colorimetric)* is a complete set of optimized buffers and reagents to quantify 5-mC in fresh or frozen urine samples using an inhibitory competitive immunoassay method. It is suitable for detect-ing total urinary 5-methylcytosine levels, resulting from whole body turnover or degradation of methylated DNA/RNA in urine from hu-mans and animals. The easy-to-follow steps takes less than 4 hours to complete from start to fi nish.

The MethylFlash™ 5-Formylcytosine (5-fC) DNA Quantifi cation Kit is a complete set of optimized buffers and reagents to colorimetri-cally quantify 5-formylcytosine (5-fC) in an ELISA-like format. It is suit-able for use with DNA isolated from any species (mammals, plants, fungi, bacteria, viruses, etc.) in a variety of forms including, but not limited to, cultured cells, fresh and frozen tissues, paraffi n-embedded tissues, and body fl uid samples. Fast and easy, the procedure can be completed within 3 hours and 45 minutes.

METHYLATED DNA QUANTIFICATION

5-mC standard was added into the assay wells at different concentrations and then mea-sured with the MethylFlash™ Urine 5-Methylcytosine Quan-tifi cation Kit (Cat. # P-1039).

Demonstration of high speci-fi city of 5-fC detection achieved by the MethylFlash™ 5-Form-ylcytosine (5-fC) DNA Quan-tifi cation Kit (Cat. # P-1041). Synthetic unmethylated DNA, methylated DNA, hydroxymeth-ylated DNA and 5-formylcyto-sine DNA standard were added into the assay wells at different concentrations and then mea-sured with the kit.

Hydroxymethylated DNA (ng)

Del

ta O

D

Input amount (ng)

OD 45

0 nm

5-mC standard (ng)

OD 45

0

*Also available in fl uorometric format (Cat. # P-1035)



www.epigentek.com • [email protected]

The EpiSonic™ 2000 is a multi-sample sonicator designed for DNA and chromatin shearing applications, capable of uniformly shearing between 150 bp and 20 kb. To ensure optimal performance and verify fragment sizes, we carefully examine and test each individual unit with a “white glove” approach. Our technicians perform full sonication runs as if it were in your own hands, then adjust and fi ne-tune the particular unit’s protocol to achieve ideal target fragment sizes. Because in the end, results are what really matter.

Features At-A-GlanceThe EpiSonic™ 2000 incorporates Digitally Adaptive Sonocavitation™ (DAS) technology to effi ciently process DNA and chromatin samples, with the following key features:

• Simple input and controls• Simultaneous sonication of up

to 12 samples• 10 min to shear 12 samples to

300 bp (50 sec/sample)• Closed vessel processing

• Reproducible and consistent fragments

• Easy installation and maintenance

• Continuous cooling system• 20-month warranty

Demonstration of consistent fragments achieved with a single run of 12-sample mul-tiplex shearing with the EpiSonic™ 2000 Sonication System.

EpiSonicmulti-functional bioprocessor

TM

Individually tested. Universally effective.

EpiSonic 2000 Sonication SystemCat. # EQC-2000

FitAmp Plasma/Serum DNA Isolation Kit Cat. # P-1004

FitAmp General Tissue Section DNA Isolation KitCat. # P-1003

Isolat

ed D

NA (n

g)

DNA amount in serum (ng)

The FitAmp™ Plasma /Serum DNA Isolation Kit (Cat. # P-1004) simply applies our proprietary DNA isolation buffer to plasma or serum. After treatment with DNA digestion buffer, the DNA is easily recovered in 8-20 µl by our specially designed F-Spin Column. DNA is ready for downstream application.

The FitAmp™ Plasma/Serum DNA Isolation Kit is a complete set of essential components which enables the experimenter to effi ciently isolate DNA from plasma, serum, and body fl uids within 12 minutes with consistent results.

DNA ISOLATION

The FitAmp™ General Tissue Section DNA Isolation Kit is a complete set of essential components which enables the ex-perimenter to effi ciently isolate DNA in any targeted microscopic tissue area on a slide. The entire procedure can be completed within 2 hours, depending on sample types, with consistent isolation conditions and produces far superior results than any competitor kits.

FitAmp Blood and Cultured Cell DNA Extraction KitCat. # P-1018

FitAmp Urine DNA Isolation KitCat. # P-1017

DNA

yield

(ug)

Cell number (x 1000)

Genomic DNA was isolated from MCF-7 cell line using the FitAmp™ Blood and Cultured Cell DNA Ex-traction Kit (Cat. # P-1018). The isolated DNA yield was quantifi ed by real time PCR.

The FitAmp™ Blood and Cultured Cell DNA Extraction Kit is a complete set of essential components which enables the experimenter to effi ciently isolate DNA from blood leukocytes and cultured mammalian cells within 20 minutes with consistent isolation conditions.

The FitAmp™ Urine DNA Isolation Kit is a complete set of essential components which enables the experimenter to effi -ciently isolate DNA from urine within 20 minutes with consistent isolation conditions.

3www.epigentek.com • [email protected]

Product Name Methylamp DNA Modifi cation Kit

BisulFlash DNA Bisulfi te Conversion

Easy Kit

BisulFlash DNA Modifi cation Kit

Methylamp Whole Cell Bisulfi te

Modifi cation Kit

BisulFlash DNA Bisulfi te Conversion

Mag-96 KitCat. # P-1001 P-1054 P-1026 P-1016 P-1050

Codename “Classic” “Easy” “Fast” “Direct” “High Throughput”

PurposeChemical-based

DNA bisulfi te conversion

Simplifi ed heat-based DNA bisulfi te

conversion

Fast heat-based DNA bisulfi te conversion

Directly convert from cells, tissues, saliva,

urine, or blood

Magnetic bead-based 96-well format bisulfi te

conversion

Key Points

• Illumina workfl ow compatible

• Most robust and reliable

• Ideal for bisulfi te sequencing, NGS, and various MSP


• Denaturation by thermalcycler

• Liquid bisulfi te con-version reagent

• Extremely fast• Ideal for real-time

MSP• Optional

sequencing protocol

• Eliminates separate DNA isolation step

• Ideal for samples containing low quantities of DNA


• Streamlined high throughput process

• Designed for large scale or automated conversions

Format Spin Column Spin Column Spin Column Spin Column 96-Well Plate

Starting Material DNA DNA DNA cells, tissue, blood DNA

Lowest Input Amount 50 pg 100 pg 200 pg 100 cells, 1 µl blood < 10 ng

Conversion Effi ciency 99.9% 99.9% 99.9% 99.9% 99.9%

Recovery >75% >75% >75% >85% >85%

Time < 2 hours 1 hour 30 minutes < 3 hours < 1 hour 20 minutes

BISULFITE CONVERSION

BisulFlash DNA Modifi cation KitCat. # P-1026

The BisulFlash™ DNA Modifi cation Kit is a complete set of opti-mized buffers and reagents for an ultra-fast DNA bisulfi te conversion. DNA denaturation and bisulfi te conversion are processed simultane-ously, reducing the entire procedure to only 30 minutes. Each reac-tion with the BisulFlash™ kit can use 0.2 ng – 1 µg of DNA. Further-more, it prevents more than 90% of DNA loss, completely converting unmethylated cytosine into uracil (>99.99%).

50 ng of genomic DNA m e t h y l a t e d in all CpG sites by DNA methy lases was treated with the Bisul-Flash™ DNA

Modifi cation Kit (Cat. # P-1026). Converted DNA was amplifi ed by real time qPCR using primers for multiple promoters containing numerous CpG sites and then directly sequenced.

Get fast bisulfi te treatment of DNA via magnetic beads and micro-plates using our BisulFlash™ DNA Bisulfi te Conversion Mag-96 Kit (Cat. # P-1050). Or, use our spin column format BisulFlash™ DNA Bisulfi te Conversion Easy Kit (Cat. # P-1054) which comes with a unique ready-to-use liquid conversion reagent to generate bi-sulfi te-converted DNA. Both BisulFlash™ kits can be fi nished in less than 1 hour and 20 minutes and completely convert unmethylated cytosine into uracil (>99.99%).

BisulFlash DNA Bisulfi te Conversion Mag-96 Kit & Easy KitCat. # P-1050 & Cat. # P-1054

Different amounts of human genomic DNA were converted using the BisulFlash DNA Bi-sulfi te Conversion Mag-96 Kit (Cat. # P-1050).

Methylamp DNA Modifi cation Kit Cat. # P-1001

The Methylamp™ DNA Modifi cation Kit is a complete set of opti-mized reagents designed to convert DNA so that methylated cyto-sines can be detected at base resolution in downstream applications, including MSP and bisulfi te-sequencing. The kit, which requires as little as 50 pg of input DNA, can be fi nished in under 2 hours and is highly compatible with Illumina sequencing workfl ows.

Different amounts of DNA isolated from a serum sample were chemically modifi ed using the Methylamp™ DNA Modifi cation Kit (Cat. # P-1001). Real time PCR was then performed using a pair of primers (β-actin) and a probe de-signed to amplify converted DNA.

Amount of starting DNA (ng)

Cycle

s


METHYLATED DNA IMMUNOPRECIPITATION

EpiQuik MeDIP Ultra KitCat. # P-1052

EpiQuik hMeDIP KitCat. # P-1038

Methylamp and EpiQuik MeDIP KitsCat. # P-1015, Cat. # P-2019 & Cat. # P-2020

The EpiQuik™ MeDIP Ultra Kit is a complete set of optimized re-agents to enrich and capture methylated DNA fragments in a conve-nient microplate-based format. A further refi nement of the predeces-sor Methylamp™ MeDIP Kit, it signifi cantly improves sensitivity and specifi city while reducing background signals, making it extremely suitable for MeDIP-Seq.

• Extremely fast and convenient 3 hour procedure

• Optimized buffers to minimize background

• Highly specifi c 5-mC Ab included for >99% specifi city

• Flexible strip-well microplate format for manual or high throughput

• Low DNA input requirement as low as 50 ng

• High reproducibility using pre-optimized MeDIP conditions

• Compatible with various downstream analysis workfl ows

The EpiQuik™ Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit is a complete set of optimized buffers and reagents to carry out a successful hMeDIP procedure for the selective enrich-ment of DNA fragments containing 5-hydroxymethylcytosine (5-hmC) in a high throughput format using DNA isolated from any species. The hydroxymethylated DNA that is enriched with this kit can be used in various downstream applications including PCR (hMeDIP-PCR) and microarrays (hMeDIP-chip) for investigating gene-specifi c hydroxy-methylation in cells or tissues. With an extremely fast and convenient protocol, the total procedure time (from input sample to ready-to-use hydroxymethylated DNA) takes less than 3 hours.

The Methylamp™ Methylated DNA Capture (MeDIP) Kit (Cat. # P-1015) starting from DNA, EpiQuik™ Methylated DNA Immuno-precipitation Kit (Cat. # P-2019) starting from cells, and EpiQuik™ Tissue Methylated DNA Immunoprecipitation Kit (Cat. # P-2020) starting from tissue, each come as a complete set of optimized buf-fers and reagents that allows the experimenter to enrich methylated DNA in a convenient 96-well plate format. The entire procedure can be fi nished in 3 to 4 hours, producing enriched methylated DNA suit-able for standardized DNA detection methods including PCR, micro-arrays, and sequencing.

• Highly effi cient enrichment of methylated DNA

• Extremely fast procedure, which can be fi nished in 3 to 4 hours

• Flexible strip microplate format for manual or high throughput• Columns for DNA purifi cation are included, saving time and

reducing labor

• Compatible with all DNA amplifi cation-based approaches

• Simple, reliable, and consistent assay conditions

Sensitive detection of gene-specifi c methylation by MeDIP-qPCR: Fully methylated HeLa DNA (500 ng) was fragmented to 100-500 bps using an EpiSonic™ 1100. The fragmented DNA was used for methyl-ated DNA enrichment with the EpiQuik™ MeDIP Kit (Cat. # P-1052). Eluted DNA was analyzed by real time PCR with primers specifi c for MLH1 sequences in the promoter regions. Results show the specifi city of the 5-mC antibody and low background for non-immune IgG. Fold-en-richment represents the amount of recovered DNA and was calculated based on the real time PCR Ct value.

Sensitive detection of gene-specifi c hydroxymethylation by hMeDIP-PCR: Human brain DNA (500 ng) was fragmented to 200-600 bps with an EpiSon-ic™ sonicator. The fragmented DNA was used for hydroxymeth-ylated DNA enrichment with the EpiQuik™ hMeDIP Kit (Cat. # P-1038). Eluted DNA was analyzed by real time PCR with primers specifi cally for OCT4 or GAPDH sequences in the pro-

moter regions. Results show that the promoter region is hydroxymethyl-ated in OCT4 but not in GAPDH. Fold-enrichment represents the amount of recovered DNA and was calculated based on the Ct value.

Enrichment of methylated DNA using the Methylamp™ Methylated DNA Capture Kit (Cat. # P-1015). 0.5 µg of DNA isolated from MCF-7 cells was added into the wells, followed by: (a) methylated DNA captured by a 5-mC antibody; (b) captured methylated DNA used for analyzing methylation levels of GAPDH and MLH1 promoters with the use of prim-ers and probes specifi c to GAPDH and MLH1 promoters, respectively.

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GAPDH MLH1

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PCR & NGS ANALYSIS

EpiNext DNA Purifi cation HT SystemCat. # P-1063

Methylamp MS-qPCR Fast Kit Cat. # P-1028

The Methylamp™ MS-qPCR Fast Kit is a complete set of essential components, which enables the experimenter to perform fast, specif-ic, sensitive, and reproducible methylation-specifi c quantitative PCR. This kit is suitable for quantitative methylation-specifi c PCR in a fast format using very minute amounts of DNA. The novel hot start DNA polymerase contained in this kit can reduce the overall time required for MS-qPCR from approximately 2.5 hours to less than 70 minutes.

MS-PCR amplifi cation of a gene region in euchromatin with internal control primers. Suc-cessful GpC methylation and bisulfi te conversion were carried out using the EpiQuik™ Chroma-tin Accessibility & Methylome Se-quencing (CAMe-Seq) Kit (Cat. # P-1048) and PCR was per-formed using Methylamp™ MS-qPCR Fast Kit (Cat. # P-1028).

EpiNext High-Sensitivity DNA Library Preparation Kit Cat. # P-1053

The EpiNext™ High-Sensitivity DNA Library Preparation Kit (Illu-mina) is a complete set of optimized reagents required at each step of the workfl ow to carry out a successful DNA library preparation. This kit is suitable for preparing a DNA library from just 0.2 ng of DNA input for Illumina-based next generation sequencing applications, includ-ing bisulfi te-seq, ChIP-seq, and MeDIP-seq. This allows limited or diffi cult samples such as tissue biopsy or embryonic samples to be viable for sequencing. The procedure, from fragmented DNA to size selection, is less than 1 hour and 30 minutes.

Size distribution of library fragments. Human placenta DNA was sheared to around 300 bps in peak size and 0.2 ng of DNA was used for DNA library preparation using the EpiNext™ High-Sensitivity DNA Library Preparation Kit (Cat. # P-1053).

EpiNext Post-Bisulfi te DNA Library Preparation Kit Cat. # P-1055

The EpiNext™ Post-Bisulfi te DNA Library Preparation Kit (Illumi-na) is a complete set of optimized reagents to prepare a DNA library -- after a successful bisulfi te conversion -- for various Illumina plat-form-based bisulfi te sequencing (bisulfi te-seq) assays. This allows for reduced fragment loss and minimized selection bias from just 1 ng of pre-bisulfi te treated input DNA. The entire procedure, from input starting material to library amplifi cation, takes only 5 hours.

Size distribution of library fragments as demonstrated by a post-bisulfi te DNA library con-structed using the EpiNext™ Post-Bisulfi te DNA Library Preparation Kit from 10 ng of input DNA (Cat. # P-1055).

The EpiNext™ DNA Purifi cation HT System (also known as “EpiNext beads”) utilizes magnetic bead technology for high throughput DNA or PCR amplicon purifi cation and DNA size selection. The system can also be used for concentrating DNA and is suitable for selectively and consistently capturing DNA fragments or PCR amplicons that are 100 bps or larger in size (Cat. # P-1063).

*Agencourt® AMPure® is a registered trademark of Beckman Coulter, Inc.

Percent recovery of input DNA fragments by AMPure XP* beads vs. EpiNext beads.

Powerful Paramagnetic Purifi cation

*



Genome-wide, base resolution DNA methylation analysis

■ Receive publication ready results■ Complete conversion (>99.9%) and maximum

recovery of bisulfi te-converted DNA■ Low input of 0.5 ng of DNA■ High yield of the constructed DNA library and low

error rates■ Up to 50x coverage

Methyl-Seq (NGS Bisulfi te Sequencing)

Expand your research capabilities with our comprehensive and accurate lab services. From epigenetic-based next-generation sequencing to compound screening and more, our staff of experts works collaboratively with you to provide the proper technical guidance and tools that will propel your research forward.

Real-Time Progress Tracker

Learn more online at:www.epigentek.com/services


dna methylation brochure 8 12 15 copy - caltag medsystems...the methylflash™ methylated dna (5-mc)...

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