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5-hmC and 5-mC Analysis with Control DNA of the Kit

DNA methylation 5-hydroxymethylcytosine HpaII

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#1 yasemin

yasemin

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Posted 31 July 2012 - 01:25 PM

Before using EpiMark® 5-hmC and 5-mC Analysis Kit with our samples, we decided to try the kit with the control DNA and control primers, but we had some problems.

First of all, we used totally 1ng (as indicated in the instruction manual of the kit) of control DNA (0.33ng of each of control DNA: methylated, hemimethylated and unmodified DNA) in DNA glucosylation reaction with 12.4ul UDP-Glucose, 31ul NEBuffer4 and nuclease-free water (total reaction volume is 310ul).

After restriction endonuclease digestion for 16 hours and proteinase K addition, we performed end-point PCR by using 10ul of 1:100 diluted restriction endonuclease digestion products by amplifying for 25 cycles as suggested in the instruction manual of the kit (total reaction volume is 50ul). Then we run the PCR products on agarose gel.

Since we use 3 different types of control DNA in equal amounts, we expect that the band of T4-BGT added and digested with HpaII sample (which identifies total methylated DNA) should be thicker than the band of T4-BGT added and digested with MspI sample (which identifies hydroxymethylated DNA). We also expect that the band of T4-BGT added and not digested sample (which identifies total amount of DNA) should be thicker than the first two bands.

However, the band of T4-BGT added and digested with HpaII sample is the thickest and the band of T4-BGT added and digested with MspI sample is the thinnest. There is no amplification in the sample in which T4-BGT is not added and MspI is added. For the samples that do not contain T4-BGT, the band of uncut sample is thicker than that of digested with HpaII as expected.

After performing real-time PCR with SYBR green and 3ul of 1:100 diluted restriction endonuclease digestion products for 40 cycles (total reaction volume is 20ul), we again could not obtain the expected results. In T4-BGT containing samples, it seems that the amount of methylated DNA is larger than the total amount of DNA present. In the samples that do not contain T4-BGT, it seems that the amount of methylated DNA is nearly the same with the total amount of DNA present.

To sum, we could not obtain the expected ratios between the samples which indicate the amounts of modified or unmodified DNAs. In addition, the main problem is about the sample that is treated with T4-BGT and digested with HpaII because it is amplified much more than expected.

What can be the cause of these problems? Is the amount of T4-BGT larger than required for 1ng of control DNA (100bp)? Do we need to perform heat inactivation for T4-BGT before adding HpaII because the result of the sample that do not contain T4-BGT but contain HpaII that identifies the same thing, which the T4-BGT+HpaII sample does, is more similar to what is expected than the result of the T4-BGT+HpaII sample. What would you recommend us to do?

Thanks in advance for your help.





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