where to buy universally methylated and unmethylated genomic DNA? - (Dec/04/2008 )
Hi all, I am new in the DNA methylation field. I am going to do some MSP on bisulfite-modified DNA. Now I am looking for a postive control and a negative control to use with the CpGenome DNA Modification Kit (Millipore). I found Millipore provides the Universal methylated and unmethylated DNA, about $240 each. It means $480 for both control. Are there better deals from other sources?
Thanks for any information!
Cheapest option is to make your own.
Take a genomic DNA sample and SssI methylaase treat it, take the same sample and whole genome amplify to make it unmethylated.
The controls from Chemicon are not entirely unmethylated we have found.
good luck
Nick
Take a genomic DNA sample and SssI methylaase treat it, take the same sample and whole genome amplify to make it unmethylated.
The controls from Chemicon are not entirely unmethylated we have found.
good luck
Nick
I also want to make the unmethylated control of mouse DNA, but our lab doesn't have the WGA kit.
It seems to be somewhat wasting to buy the kit only for this purpose in our lab.
Is there an alternative way to make unmethylated postivie control in this case?
I couldn't find any commercial 'mouse unmethylated' DNA.
Maybe I could extract gDNA from NIH3T3 cell line after treating it with DNMT inhibitor? What do you think about this?
I think you would still have some residual methylation there.
It also depends on what you want to look at in the end, if you are only looking at one or two loci, it maybe best to just PCR amplify the regions you are interested in and take some as the unmethylated control while the rest you SssI methylate and use as 100% methylated DNA and then mix different proportions of each.
N
I think you would still have some residual methylation there.
It also depends on what you want to look at in the end, if you are only looking at one or two loci, it maybe best to just PCR amplify the regions you are interested in and take some as the unmethylated control while the rest you SssI methylate and use as 100% methylated DNA and then mix different proportions of each.
N
Thank you so much! :-)
Then, maybe I should just PCR. Could you tell me a little more in detail?
How long a segment do you usually amplify for the purpose above?
How would I design the primer?
and can I just use the PCR product for subsequent bisulfite treatment and MSP after phenol/chloroform extraction?
You can also do a PCR reaction with 5-methyl dCTP instead of dCTP in the PCR reaction. This will also methylate at sites other than CG sites. The denaturing temperature of 5-m dCTP is higher than normal DNA, so you might want to raise the PCR denaturing temperature.
How long a segment do you usually amplify for the purpose above?
How would I design the primer?
and can I just use the PCR product for subsequent bisulfite treatment and MSP after phenol/chloroform extraction?
Hi awhite0,
You can just amplify the region of interest that you want to make a standard curve for, so it can be 1-2kbp and just design normal genomic DNA primers for your region of interest.
Now the complexity of your amplicon is far less than that of genomic DNA and this should be taken into account but you can certainly use it for bisulphite treatment. So you can also clone your amplicon into a sequencing vector and then methylate that then bisulphite treat. We clone into a methylation negative E. Coli!!!
good luck!
@ phage, that's a good idea with the PCR!! Any particular Taq that needs to be used, is it like incorporating other labelled NTP's?
Nick
See this article:
PCR with 5-methyl-dCTP replacing dCTP.
Wong KK, McClelland M.
Nucleic Acids Res. 1991 Mar 11;19(5):1081-5.
PMID: 1850509