Zymo Research는 1994년도에 설립된 미국 회사로 고품질 키트 제조회사 입니다.
각종 샘플(세포, 조직, 환경샘플 등)로부터 고품질
DNA나 RNA를 가장 쉽고 빠르게 뽑을 수 있습니다.
이 외에도, 각종 Epigenetics 관련 제품들
(DNA Methylation kit 등)과 Microbiomics
(샘플 채집부터 분석까지의 전 단계의 제품)
연관 제품들이 준비되어 있습니다.
Highlights
For high-throughput detection of global 5-methylcytosine (5-mC) in DNA
The streamlined workflow can be completed in less than 3 hours
서한형 대리
Zymo Research 제품 담당자
경신과학(주)
영업부
H.P) 010-8832-6303
HanHyung Seo
Zymo Research Brand Manager
Kyongshin scientific Co., Ltd.
Sales Department
H.P) 82)10-8832-6303
제품소개
Highlights
For high-throughput detection of global 5-methylcytosine (5-mC) in DNA
The streamlined workflow can be completed in less than 3 hours
Product Description
The 5-mC DNA ELISA Kit allows researchers to accurately quantitate 5-mC in any DNA sample in less than 3 hours. The kit features an Anti-5-Methylcytosine monoclonal antibody that is both sensitive and specific for 5-mC. The assay is compatible with a wide range of input DNA from vertebrate, plant, and microbial sources as well as methylated PCR amplicons and fragmented DNA. Percent 5-mC in a DNA sample can be accurately quantified from a standard curve generated with specially designed controls included with the kit. Also, the fast, streamlined workflow is ideal for high-throughput analyses.
Technical Specifications
Detection
> 0.5% 5-mC 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 input DNA/well. Compatible with DNA in the range of 10-200 ng/well.
Sample Source
Purified genomic DNA, plasmid DNA, PCR amplification products, or DNA fragments in water, Tris-EDTA, or similar.
Make sure the correct buffers were used to coat, block and wash the plate. Verify that HRP developer and the Secondary Antibody were stored and prepared correctly.
Generate a standard curve with absorbances of control DNAs using a logarithmic curve y= ln(x)+b.
Calculate the %5-mC for each sample using the standard curve equation. To solve the equation, determine the value of x (%5-mC) using e(y-b/m).
To determine the percent CpG methylation (%5mC/CpG sites) of your samples (depends on sample origin), multiply the observed %5-mC from the standard curve by the fold difference between E. coli (control DNA derives from E. coli), and your genome of interest (see table below). You can stop here and report your data as %CpG methylation.
Calculate global methylation (%5mC/total cytosine) by multiplying the calculated %CpG methylation by the total cytosine/genome length.
E. coli
Total genome [bp]: 4.639.675
CpG site: 346.670
Total Cytosines: 1.179.554
CpG/genome length (CpG density): 0,07472
Fold difference CpG density (Ecoli/OOI): 1,000
Cytosine / genome length: 0,254
Mouse
Total genome [bp]: 2.654.895.218
CpG site: 21.342.492
Total Cytosines: 534.142.064
CpG/genome length (CpG density): 0,00804
Fold difference CpG density (Ecoli/OOI): 9,295
Cytosine / genome length: 0,201
Potato
Total genome [bp]: 705.934.411
CpG site: 10.823.234
Total Cytosines: 115.562.590
CpG/genome length (CpG density): 0,01533
Fold difference CpG density (Ecoli/OOI): 4,873
Cytosine / genome length: 0,164
Human hg19
Total genome [bp]: 3.137.161.264
CpG site: 28.700.086
Total Cytosines: 585.012.752
CpG/genome length (CpG density): 0,00915
Fold difference CpG density (Ecoli/OOI): 8,167
Cytosine / genome length: 0,186
Rat rn4
Total genome [bp]: 2.834.127.293
CpG site: 24.799.282
Total Cytosines: 572.296.669
CpG/genome length (CpG density): 0,00875
Fold difference CpG density (Ecoli/OOI): 8,539
Cytosine / genome length: 0,202
Zebrafish danRer7
Total genome [bp]: 1.412.464.843
CpG site: 25.251.167
Total Cytosines: 258.711.448
CpG/genome length (CpG density): 0,01788
Fold difference CpG density (Ecoli/OOI): 4,180
Cytosine / genome length: 0,183
Pig
Total genome [bp]: 2.808.525.991
CpG site: 30.460.512
Total Cytosines: 525.183.245
CpG/genome length (CpG density): 0,01085
Fold difference CpG density (Ecoli/OOI): 6,889
Cytosine / genome length: 0,187
Rabbit
Total genome [bp]: 2.737.490.501
CpG site: 36.000.387
Total Cytosines: 569.377.656
CpG/genome length (CpG density): 0,01315
Fold difference CpG density (Ecoli/OOI): 5,682
Cytosine / genome length: 0,208
Example: From the created standard curve, the calculate %5mC of your mouse sample is 2.5%. Determine %5mC/CpG sites: multiply %5mC by the fold difference in the CpG/genome length (see table, organism mouse) = 2.5 x 9.3=23.25 % CpG methylation. Determine (%5mC/total cytosine= global methylation): multiply the calculated %5mC/CpG by 0.201= 4.67% global 5mC.
Yes, the control DNAs can be adjusted to be assayed for lower 5-mC percentages (i.e. 0, 2.5, 5, 2.5, 10%, etc.). The kit can accurately detect 5-mC down to 0.5%. We recommend including a 0% standard where the measured O.D. represents the background absorbance. If absorbance of the samples is above the 0% control, you can extrapolate the % 5mC from the standard curve.
Components will ship on blue ice and dry ice. Upon arrival make sure to store buffers at 4°C, Control DNA and Anti-5-mC antibody at -20°C. Secondary antibody can be stored at 4°C for 1 week; for long-term storage prepare small aliquots and keep at -20°C.
The kit is optimized for 100 ng/well however you can use down to 25 ng/well. The sample DNA should be assayed at the same concentration as the standards.
