5-Carboxylcytosine DNA Standard
Catalog No: 55014
Format: 0.5 µg

Quality Control: 5-Carboxylcytosine DNA Standard Kit is quality control tested
by dot blot using Active Motif’s 5-Carboxylcytosine antibody (Catalog No.
61225) at a 1:2,000 dilution with an overnight incubation. The 5Carboxylcytosine DNA standard is tested alongside the equivalent Unmodified
DNA standard. The 5-Carboxylcytosine DNA standard is specificallyrecognized by the antibody and there is no cross-reactivity for other DNA
modifications or the Unmodified standard (Figure 1).
Contents:
5-Carboxylcytosine DNA standard, 0.5 µg (50 ng/µl)
Unmodified DNA standard, 0.5 µg (50 ng/µl)
The 5-Carboxylcytosine DNA standard consists of a 38 bp double-stranded
DNA oligonucleotide. The forward strand contains 8 carboxylcytosine DNA
modifications, while the reverse strand contains 4 carboxylcytosine DNA
modifications. The annealed dsDNA therefore contains a total of 12
carboxylcytosine residues. The Unmodified DNA standard is the equivalent
dsDNA sequence without any cytosine modifications and can be used as anegative control.

Figure 1:
Varying amounts of dsDNA from theMethylated DNA Standard Kit (Catalog No.
55008) or 5-Carboxylcytosine DNA Standard
Kit were spotted onto a nylon membrane andprobed with 5-Carboxylcytosine antibody(Catalog No. 61225, 1:2000 dilution).

Forward Strand: 5’ -AGCCXGXGCXGXGCXGGTXGAGXGGCXGCTCCCGCAGC- 3’
Reverse Strand: 3’ -TCGGGCGCGGCGXGGXCAGXTCGXCGGCGAGGGCGTCG- 5’
X indicates a carboxylcytosine residue in the 5-Carboxylcytosine standard
X indicates a cytosine residue in the Unmodified DNA standard
Dot Blot Protocol
1. Prepare the necessary dilutions of the 5-Carboxylcytosine DNA standards.
We recommend between 3 ng - 50 ng.
2. For detection of ssDNA, denature the DNA standards with heat, 95°C for
10 minutes, immediately followed by transferring the denatured DNAstandards to ice.
3. Spot 1 µl of each DNA dilution onto a positively charged nylon membrane.
Allow the membrane to dry 5-10 minutes.
4. Block the membrane with TBST (10 mM Tris, 150 mM NaCl,0.1% Tween-20) and 5% non-fat milk powder for 1 hour with rotation.
5. Dilute 5-Carboxylcytosine pAb (Catalog No. 61225) 1:2000 in TBST + 5%non-fat milk powder. Ensure that the entire membrane is covered with theantibody solution.
6. Incubate overnight at 4°C.
7. Wash the membrane 3 times with TBST, 10 minutes per wash.

Figure 2:
Comparison of single-stranded DNA (ss) anddouble-stranded DNA (ds) from the 5Carboxylcytosine DNA Standard Kit by dot blot
with a 1:2000 dilution of 5-Carboxylcytosine Ab(Cat. No. 61225).

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Quality Control:
Dot Blot Protocol continued
8. Incubate with HRP-conjugated anti-rabbit IgG secondary antibody diluted
1:2000 in TBST + 5% non-fat milk powder for 1 hour at room temperature.
9. Wash the membrane 3 times with TBST, 10 minutes per wash.
10. Develop with ECL reagents according to manufacturer’s instructions.
Optimize exposure time as needed.
Storage and Guarantee: DNA standards should be stored at -20°C. This
product is guaranteed stable for 6 months from date of receipt when stored
properly.

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