Protocol for DNA Methylation analysis using restriction enzyme digest - (Nov/04/2006 )

this is the first time to do the DNA Methylation analysis by restriction enzyme digestion(southern blot), I have no the protocol. could you eyery body here send me the protocol in details ?
for example: volume of enzyme, gel length ,volts, and so on, thank you very much!
my email:
fujunqin@hotmail.com

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qinfujin,

there are a lot of parameters you have stated that vary according to what you are assaying for, so could you please share with us what you intend to look at for methylation? Single copy gene? Repeat elements? centromere sequences? telomere sequences?

Nick

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I intend to check whether my transgenic plants(oryza sativa) cause the change of DNA Methylation in 5srDNA, centromere sequences and telomere sequences?
otherwise, I will check the DNA methylation of some transposon(multi-copy gene)?
my first time to do this , so need your help!

thank you !

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hi qinjufin,

There are many papers in the literature that perform such an assay.

I would digest with the classical pair of RE HpaII/MspI giving you methylation sensitive and insensitive digests respectively. To detect single copy genes as much as 10ug of gDNA is required for each lane, because you are intending to look at high copy number sequences, you can get away with as little as 1-5ug to start with. To ensure digestion, I digest with an excess of RE overnight and typically run the digests on a 30cm 1% agarose gel at 1-3V/cm for the entire day before I go on to blot onto Hybond N nylon membranes overnight in an alkali transfer buffer.

I follow the protocols outlined in "The Bible" known as Molecular Cloning by Sambrook and others, that would get you in good steed.

Nick

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thank you very much, nick!
I have some others question( my question is too simple, maybe)
1. how much of the RE (NEB) ? Because there are too much site of the RE (HpaII/MspI ), how to make sure the excess of RE !
2. is the gel TAE or TBE ?
3. IS the gel must be 30cm? what about 20cm ?
4.whether can use the Neutral transfer buffer?

qinfujun

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To ensure full digestion, I use 50-100U of enzyme (we have the concentrated stocks) for 10ug of gDNA

2. gel can be either TAE or TBE
3. we have 30cm gels, so 20cm would be just as good, you would lose a little resolution though i would say.
4. yes you can. alkali ensure denaturation of your DNA to single strands though so the probe is mmore likely to hyb.

good luck!

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thanks for nick's kindly help!

how much the volume of digestion, 20ul or 50ul(after digestion, concentrate it)?

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as the combs for our agarose gels can only fit at most 40ul, I digest in 30ul.

Nick

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thank you ! nick!
because of the time difference,wait for your post need at least 12hrs.
today, I do the digestion with reaction volume about 50μl, tomorrow I will reduce the sample volume to about 20-30 μl in the vacuum concentrator, is it ok?
in addition, because my tray is not big enough for 30cm long gel, only for 25cm at most!

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25cm should give you a good separation.

good luck!

Nick

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