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How to create a fully methylated control?


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4 replies to this topic

#1 Mimoza

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Posted 12 March 2013 - 03:59 AM

Most people are using the methyltranferase M.SssI to creative fully methylated controls. However, from what I have read, M.SssI methylates only CpG sites. Thus, the rest of Cs of the genome which are not followed by a G remain unmethylated (or have random methylation status depending on the sample). So, this is not a fully methylated control.
Do you know any way to have a truly completely methylated control, independently from the species?
Thanks a lot,
Mimoza

#2 lmg

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Posted 12 March 2013 - 04:09 AM

You buy one from Qiagen (human). The prize is worth the effort.

best,
lmg

Edited by lmg, 12 March 2013 - 04:10 AM.


#3 Mimoza

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Posted 12 March 2013 - 04:18 AM

Thanks for the answer.
I'm not working with human and I need to have a DNA from my species (2 different fish). Any other idea?

#4 phage434

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Posted 12 March 2013 - 10:15 AM

You can PCR with 5-me dCTP instead of dCTP in the reaction. You'll have to mix with dATP, dGTP, and dTTP yourself.

#5 et2b

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Posted 25 August 2013 - 11:27 PM

Hi, most methylated DNAs from commercial parties are not entirely methylated. 

A friendly lab once tried with replacing the Cytosine base in dNTP with 5-methylcytosine and doing a whole genome amplification reaction. 

the are still using it and generally happy. 

Also an excelent way to make fully unmethyled DNA, do a whole genome amplification using normal cytosine when making your dNTP mixture.

 

the kit they used was officially for human but it worked for their insect species. maybe order a sample at a supplyer and give it a go? 






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