hMeDIP Kit Overview
The hMeDIP Kit is designed to immunoprecipitate and enrich for DNA fragments containing 5-hydroxymethylcytosine (5-hmC). The hMeDIP Kit contains a highly specific purified 5-hmC antibody and the necessary buffers to perform methylated DNA immunoprecipitation (MeDIP). The affinity of the antibody enables separation of 5-hmC DNA from 5-mC DNA. Additionally, the antibody works to efficiently immunoprecipitate either double-stranded or single-stranded input DNA.
Active Motif's fast, magnetic protocol has been streamlined to minimize the number of wash and incubation steps, saving you valuable time. The kit also includes unmethylated, 5-methylcytosine and 5-hydroxymethylcytosine DNA controls and PCR primers that can be used to verify the efficiency of the enrichment.
hMeDIP Kit Highlights
- Specific antibody only enriches 5-hmC methylated DNA
- Works with either double-stranded or single-stranded DNA
- Optimized magnetic protocol to minimize hands-on time
- Includes unmethylated, 5-methylcytosine and 5-hydroxymethylcytosine control DNAs and PCR primers to verify efficiency
hMeDIP Kit Contents
*Protein G magnetic beads are shipped on dry ice and will arrive frozen. DO NOT refreeze the magnetic beads after their first use. Once thawed, the Protein G magnetic beads should be stored at 4°C.
- 5-hydroxymethylcytosine pAb (1 μg/μl), 50 μg, Store at -20°C
- Rabbit IgG (1 μg/μl), 100 μl, Store at -20°C
- Protease Inhibitor Cocktail (PIC), 100 μl. Store at -20°C
- Buffer C, 10 ml, Store at -20°C
- Buffer D, 10 ml, Store at -20°C
- Elution Buffer AM2, 1.6 ml, Store at -20°C
- Neutralization Buffer, 1.6 ml, Store at -20°C
- Hydroxymethylated APC DNA (50 ng/μl) 10 μl, Store at -20°C
- Methylated APC DNA (50 ng/μl) 10 μl, Store at -20°C
- Unmethylated APC DNA (50 ng/μl) 10 μl, Store at -20°C
- APC PCR Primer Mix (2.5 μM) 400 μl, Store at -20°C
- Protein G magnetic beads* 250 μl, Store at 4°C
- 0.2 ml PCR tubes 1 pack, Store at RT
- Bar magnet and glue dots, Store at RT
hMeDIP Kit Data
hMeDIP Kit FAQs
How should I prepare libraries for hMeDIP-seq?
The antibody used for hMeDIP works with double-stranded fragments, and so the immunoprecipitation can be done with dsDNA. Any library prep protocol designed for sub-nanomolar levels double-stranded DNA, should work. Our recommendation is to use the NEBNext® Ultra™ II DNA Library Prep with Sample Purification Beads.
What are the “spiked” controls used for in the hMeDIP Kit?
The controls consist of a 338 base pair fragment containing 122 cytosine residues from the APC (adenomatosis polyposis coli) gene promoter. The unmethylated, 5-methylcytosine and 5-hydroxymethylcytosine containing versions were created by the inclusion of either unmethylated dCTP, 5-methyl dCTP or 5-hydroxymethyl dCTP. Adding these is an optional step used to verify IP efficiency. Note that in APC locus is not methylated in normal human gDNA. Therefore the only enrichment should be from the “spiked” hMeDNA. These DNA standards are also available separately as catalog no. 55008.
Why is neutralization buffer added after IP?
The Elution Buffer AP4 has a very low pH.
Can the hMeDIP Kit be used to enrich DNA for other marks other than 5hmC?
In a publication by Li, Z., Zhao, S., Nelakanti, R.V. et al., a N6-methyladenine (n6-mA) antibody was used to immunoprecipitate DNA using the Active Motif hMeDIP Kit components, using the alternative denaturing DNA IP protocol. Please see the publication for details.
hMeDIP Kit Publications
Search our database of customer publications that have used our hMeDIP Kit.
hMeDIP Kit Documents
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Name | Format | Cat No. | Price | |
---|---|---|---|---|
hMeDIP | 10 rxns | 55010 | ¥96,000 | Buy |