Hi All,
I am starting to develop a LC-MS/MS based quantification method to determine the global DNA methylation level in cultured cells after compound treatment. I am following this paper right now (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070205/) but I wonder if there are better protocols that someone could share. Two specific questions I want to ask is:
1. DNA degradase vs DNA degradase Plus - why do I need to use the DNA degradase Plus for LC-MS/MS?
2. Are there cheaper way to make DNA standard for the methylated/non-methylated DNA than the one suggested on the paper? This 897-bp DNA standard set cost $346 for 2 ug! (https://www.zymoresearch.com/epigenetics/dna-hydroxymethylation/hydroxymethylated-dna-standards/5-methylcytosine-5-hydroxymethylcytosine-dna-standard-set)
Any suggestion will be deeply appreciated.
Thank you in advance,
Lyra
1) Degradase only results with nucleotides with the phosphate group still attached. Degradase plus goes 1 step further by removing the phosphate group to give nucleosides, which is what you want to inject into your LS-MS/MS
2) That's actually not so expensive. Furthermore, depending on your machine, you will only need pg of the standard so 2ug is plenty to last a long time.
Hi there,
totally agreed with previous answers about your two questions.
We don't have a protocol for MS but you can have a look here for conditions for RP-HPLC
Best!
Hi W.S. Sho and Conchita,
Thank you so much for your reply!
I decided to buy the standard. I will let you guys know how it goes.
Best,
Lyra
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