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methods for global methylation - what is the choice method? (Apr/16/2007 )

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QUOTE (krümelmonster @ Apr 18 2007, 02:57 PM)
Hi bunja, the mystery protocol relates to a HpaII/MspI digestion followed by a single-nucleotide extension, first described by Progribny et al. A non-radioactive version of this assay has been described by Bönsch et al 2004. It is relatively easy to do, but it surely is not as reliable as the HPLC or MS techniques. If you want, I can PM you a protocol.

Krümel



Krumelmonster,
Thanks, I don't understand why we should not use with the most reliable technique available. Ambiguous results lead to more experiments. So maybe it is time for me to go into stealth mode for this one ph34r.gif. Those experiments are more fun anyway.

Thanks
Bunja

-bunja-

QUOTE (bunja @ Apr 18 2007, 12:05 PM)
Thanks guys,
As for not wanting to do HPLC and mass spec, it is not that I don't want to do it, I am ready to go, it is more convincing my boss that is the way to go. He keeps saying that there is some other enzymatic way to do it without producing a reference for a protocol. As for an ELISA and blot method, from my experience doing blots, they are not quantitative enough, colorometric and chemolumenescence are not good for actual quantitation. I know that infrared like a Lycor scanner can be quantitative, but I don't think it is the way to go.

I suppose I am looking for this mystery protocol that my boss is talking about ph34r.gif.

Thanks

I have done the dot blot using the infrared scanner. To me, it is easy, but not very sensive.

-hn37041-

Hi Bunja,

I know how do you feel, because I have the same problem in my lab. I know that there are many methods to measure global methylation but my boss insist in the radiolabel method (using HpaII and MspI). I'm having o lot of difficult in this protocol because the duplicates don't match.
Could anyone send me more details about this method?

Thanks a lot
Anna

-brstudent-

QUOTE (krümelmonster @ Apr 18 2007, 11:57 PM)
Hi bunja, the mystery protocol relates to a HpaII/MspI digestion followed by a single-nucleotide extension, first described by Progribny et al. A non-radioactive version of this assay has been described by Bönsch et al 2004. It is relatively easy to do, but it surely is not as reliable as the HPLC or MS techniques. If you want, I can PM you a protocol.

Krümel


Hi Krümel,

I looked up the publication by Bönsch et al. I might try it, but have some questions left. Do you have experience with it or could you send or upload a protocol perhaps? The following things are not clear to me:
* Is there a sort of purification step necessary between the digestion and the nucleotide extension step?
* 0.4 mM Cy5-dCTP in 25 µL means 10 nmol of Cy5-dCTP necessary, which is almost half of a 25 nmol tube from Amersham. This seems to be an expensive thing for analyzing multiple samples...
* How is the fluorescence measured? Is it done in a multiwell plate with the 100 µL of eluted labelled DNA?

Michiel

-MichielV-

QUOTE (MichielV)
I looked up the publication by Bönsch et al. I might try it, but have some questions left. Do you have experience with it or could you send or upload a protocol perhaps? The following things are not clear to me:
* Is there a sort of purification step necessary between the digestion and the nucleotide extension step?

No, we just use 20 µl pf the digested DNA.
QUOTE (MichielV)
* 0.4 mM Cy5-dCTP in 25 µL means 10 nmol of Cy5-dCTP necessary, which is almost half of a 25 nmol tube from Amersham. This seems to be an expensive thing for analyzing multiple samples...

Well, it is not exactly cheap. But I think, this is a mistake in the paper. We use the 25nmol tube, dilute 1:50 with H2O and take 2µl per 50µl reaction.
QUOTE (MichielV)
* How is the fluorescence measured? Is it done in a multiwell plate with the 100 µL of eluted labelled DNA?

Yes, exactly. We use a Biorad Photometer for that purpose.

Hope that helps,

Krümel

-krümelmonster-

QUOTE (krümelmonster @ May 10 2007, 05:12 PM)
Hope that helps,

Krümel


Hi Krümel,

Thanks. It makes a big difference between 2.5 reactions per tube and 625 reactions per tube of Cy5-dCTP!

You work in a 50 µL reaction volume for the nucleotide extension, instead of the 25 µL from the paper.
I assume you upscale the polymerase concentration, resulting in the following components of the mixture:

* 20µL of digested DNA (approx. 0.5-1 µg)
* 4 units of polymerase (= approx. 1 µL of a 5u/µL tube)
* 5 µL of 10x polymerase buffer (if necessary, additional MgCl2)
* 2 µL of 1:50 diluted 1 mM Cy5-dCTP

Do you fill the rest of the volume with water? Please correct me if I'm wrong...

About the fluorescence, do you think it would work on a Perkin-Elmer LS50B spectrofluorometer?

-MichielV-

Hi MichielV,

you're right, we scaled the whole reaction up and fill to 50µl with water. I think your spectrofluorometer should be fine, also.
K.

-krümelmonster-

QUOTE (krümelmonster @ May 11 2007, 11:22 AM)
Hi MichielV,

you're right, we scaled the whole reaction up and fill to 50µl with water. I think your spectrofluorometer should be fine, also.
K.


Thanks again K.

One more question though: I checked the spectrofluorometer and the emission multiplier goes up to 650 nm, which is too low for cy5. But I guess the procedure should also work with cy3-labeled dCTP?

-MichielV-

I'd guess so.

-krümelmonster-

Hi,

I tried fluorescence measurements on a fujifilm FLA-5100. However I got quite high autofluorescence from my multiwell plate.
What type of plates did you use?

Michiel

-MichielV-

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