Hi there!
Like many of you, I´m also starting to make methylation spesific PCR, and I would like to know what is the best way to get totally methylated human DNA as a positive control for my PCRs?
I guess the negative control would be human sperm DNA.
Yours, Daisy
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#2
pcrman
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Posted 17 September 2004 - 04:52 PM
To get fully methylated DNA as a positive control, you can treat plasimd DNA with SssI methyltransferase which catalyzes de novo methylation on CpG sites. You can also purchase such DNA from commercial sources such as Chemicon.
#3
genie
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Posted 19 July 2011 - 05:12 PM
pcrman, on 17 September 2004 - 04:52 PM, said:
To get fully methylated DNA as a positive control, you can treat plasimd DNA with SssI methyltransferase which catalyzes de novo methylation on CpG sites. You can also purchase such DNA from commercial sources such as Chemicon.
dear Epigenetist,
could you please put up a protocol on how to treat plasimd DNA with SssI methyltransferase.
which plasmid did you use? can i buy SssI methyltransferase from NEB? how long do you incubate for complete conversion? besides, also i would like to know your comments about the Zymo kit and Epitect kit, or do you do your bisulphite conversion manually?
please reply. thank you for your time and responses.
#4
MoB
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Posted 20 July 2011 - 01:39 AM
genie, on 19 July 2011 - 05:12 PM, said:
pcrman, on 17 September 2004 - 04:52 PM, said:
To get fully methylated DNA as a positive control, you can treat plasimd DNA with SssI methyltransferase which catalyzes de novo methylation on CpG sites. You can also purchase such DNA from commercial sources such as Chemicon.
dear Epigenetist,
could you please put up a protocol on how to treat plasimd DNA with SssI methyltransferase.
which plasmid did you use? can i buy SssI methyltransferase from NEB? how long do you incubate for complete conversion? besides, also i would like to know your comments about the Zymo kit and Epitect kit, or do you do your bisulphite conversion manually?
please reply. thank you for your time and responses.
You certainly can purchase M.SssI methyltransferase from NEB. Find a protocol and ordering informations here.
From my point of view both bisulfite conversion kits work excellent. Do not use a 'home-brew' method, but rely on the good performance of the kits and use one of their modified protocols if you have a very 'special' sample.
Hope that helps...
MoB
