Highlights
- Sensitive and specific detection of 5-hydroxymethlycytosine (5-hmC) from a variety of samples
- Ideal for global 5-hmC quantitation and high throughput screening
- Streamlined workflow can be completed in 3 hrs
Description
The Quest 5-hmC DNA ELISA Kit is both sensitive and specific and can be used to accurately detect 5-hmC DNA in a variety of samples. The kit is compatible with a wide range of input DNA including intact vertebrate, plant, and microbial genomic DNA, as well as enzyme-digested and mechanically sheared fragments. The Control DNA Set included with this kit has been calibrated to accurately quantify the percent 5-hmC in sample DNA by use of a standard curve. Also, the fast, streamlined workflow is ideal when analyzing/screening large numbers of samples.
Technical Specifications
| Detection | ≥ 0.02% 5-hmC per 100 ng DNA |
|---|---|
| Equipment | Incubator and ELISA plate reader. A multi-channel pipettor is recommended. An automated plate washer may be used for blocking and wash steps. |
| Input | This protocol is optimized for 100 ng of input DNA/well. Compatible with DNA in the range of 20-200 ng/well. |
| Sample Source | Purified genomic DNA in water, Tris-EDTA, or similar. |
Product FAQ
Q1: What is the shelf life of the ELISA kits?
We guarantee integrity of kit components for up to 6 months from date of purchase
Q2: How to store the components of the 5h-mC ELISA kit?
Shipping of components is on blue and dry ice. Upon arrival, make sure to store buffers at 4°C, Control DNA and Anti-5hmC antibody at -20°C, and the Anti-DNA antibody can be stored at -20°C for 1 week (For long-term storage keep at -80°C).
Q3: Where are the Control DNA derived from?
The DNA ELISA controls are genomic DNA. Control A is E. coli genomic DNA as it comprises 0% 5-hmC. The other controls derive from various mouse tissues and the %5-hmC is specific to each tissue. The levels of 5-hmC are were quantify by mass-spec.
Q4: Which wavelength should be used for the ELISA?
The optimal absorbance is at 405 nm, however 405-450 nm will work as well.
Q5: How to quantify the percentage of 5-hmC in a DNA sample?
To calculate global %5-hmC, control DNAs and samples must be assayed on the same plate. Use the equation x=y-b/m and solve for x (%5-hmC).
Q6: Why is there no color development after addition of HRP developer?
1. Make sure the correct buffers were used to coat, block and wash the plate.
2. Double-check that HRP developer and the Anti-DNA HRP antibody were stored and prepared correctly.
Q7: My ODs so low. What should I check?
1. Make sure that standard curve developed properly (R2 > 0.9).
2. Verify that all samples and controls are prepared correctly, mixed well before dilution and addition to the wells.
3. Ensure that anti-DNA HRP antibody was stored and prepared correctly before addition to the wells.
4. %5-hmC levels of your sample might be too low for the assay to detect (detection limit is 0.01% in 100 ng of denatured DNA).
Citations
Kit Components
| Cat # | Name | Size |
|---|---|---|
| D5425-1-15 | Coating Buffer | 15 ml |
| D5425-2-30 | 10X ELISA Buffer | 30 ml |
| D5425-3-100 | Anti-DNA HRP Antibody (100X) | 100 µl |
| D5425-4-15 | HRP Developer | 15 ml |
| D5425-5-1 | Control A (100 ng/µl) | 40 µl |
| D5425-5-2 | Control B (100 ng/µl) | 40 µl |
| D5425-5-3 | Control C (100 ng/µl) | 40 µl |
| D5425-5-4 | Control D (100 ng/µl) | 40 µl |
| D5425-5-5 | Control E (100 ng/µl) | 40 µl |
| A4001-25 | Anti-5-Hydroxymethylcytosine Polyclonal Antibody | 25 µg |
| C2020 | 96-Well ELISA Plate | 12 x 8-Well Strips |
Quest 5-hmC DNA ELISA Kit
D5425 / D5426
| Cat # | Name | Size |
|---|
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