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Methylation troubleshooting - (Sep/01/2008 )

Hi!

I am performing methylation assays, using the NEB enzymes (Msss I, and I also tested Hha I )
But I am experiencing problems of efficiency during the methylation step.

I proceed according to the protocols given with the enzymes (SAM conditions, DNA quantity, 1H at 37°C…) and still, the methylation of the CpG islands doesn’t seem to be optimal.
After the methylation I do a bisulfite treatment (that is working very well according to my controls)
Still, the sequencing of my genomic DNA shows a partial methylation. My concern is that I don’t want to methylate too long (as the next step would be to proceed on nuclei and localize nucleosomes on my locus)

Similar experiments have been done using NEB enzymes, and it was shown that 15 minutes are sufficient while 1H is not enough in my hands!

So, I would like to know if there are any steps of the protocol that I should do with special care? Could the quality of the DNA interfere with the methylation? (anway, I purified my DNA and the ratios and salts are good)
I also tested diverse concentrations, volumes and quantities…


Thank you very much for your help, smile.gif

Marie.

-marieB-

Hi Marie,

I am at home so i dont have the protocols handy, but the included instructions should tell you that with the inlcuded enzyme buffer, SssI methylase methylates DNA in a distributive manner, if you omit Mg ions from the buffer it becomes processive.

We make the buffer without magnesium and perform two rounds of methylation on our DNA to ensure complete methylation.

It may sound like you have exhausted your SAM in the reaction as well if it is not methylating even after 1 hour incubation!

Nick

-methylnick-

QUOTE (methylnick @ Sep 1 2008, 11:42 PM)
Hi Marie,

I am at home so i dont have the protocols handy, but the included instructions should tell you that with the inlcuded enzyme buffer, SssI methylase methylates DNA in a distributive manner, if you omit Mg ions from the buffer it becomes processive.

We make the buffer without magnesium and perform two rounds of methylation on our DNA to ensure complete methylation.

It may sound like you have exhausted your SAM in the reaction as well if it is not methylating even after 1 hour incubation!

Nick



Hi Nick!
Thanks a lot!
I already tried to add fresh SAM twice during the methylation process.
So the next step will be the Mg ion control. Thanks again!!
Marie.

-marieB-

QUOTE (methylnick @ Sep 1 2008, 11:42 PM)
Hi Marie,

I am at home so i dont have the protocols handy, but the included instructions should tell you that with the inlcuded enzyme buffer, SssI methylase methylates DNA in a distributive manner, if you omit Mg ions from the buffer it becomes processive.

We make the buffer without magnesium and perform two rounds of methylation on our DNA to ensure complete methylation.

It may sound like you have exhausted your SAM in the reaction as well if it is not methylating even after 1 hour incubation!

Nick


Hi Nick!
I forgot to tel you that the methylation without Mg ions is really much better!
thanks you so much!!!!
Marie.

-marieB-

QUOTE (methylnick @ Sep 1 2008, 11:42 PM)
Hi Marie,

I am at home so i dont have the protocols handy, but the included instructions should tell you that with the inlcuded enzyme buffer, SssI methylase methylates DNA in a distributive manner, if you omit Mg ions from the buffer it becomes processive.

We make the buffer without magnesium and perform two rounds of methylation on our DNA to ensure complete methylation.

It may sound like you have exhausted your SAM in the reaction as well if it is not methylating even after 1 hour incubation!

Nick


Hey Nick!

How do you test the completion of your methylation reaction (COBRA, realtime PCR, ...)? Do you think it's necessary to perform two rounds of SssI methylation?

Thanks

MoB

-MoB-

QUOTE (MoB @ Nov 5 2008, 07:34 AM)
QUOTE (methylnick @ Sep 1 2008, 11:42 PM)
Hi Marie,

I am at home so i dont have the protocols handy, but the included instructions should tell you that with the inlcuded enzyme buffer, SssI methylase methylates DNA in a distributive manner, if you omit Mg ions from the buffer it becomes processive.

We make the buffer without magnesium and perform two rounds of methylation on our DNA to ensure complete methylation.

It may sound like you have exhausted your SAM in the reaction as well if it is not methylating even after 1 hour incubation!

Nick


Hey Nick!

How do you test the completion of your methylation reaction (COBRA, realtime PCR, ...)? Do you think it's necessary to perform two rounds of SssI methylation?

Thanks

MoB



I used to treat 10ug of DNA in 500uL of reaction with 10x SAM (10 times the NEB reccomendation). I'd do 5 bisulfite treatments from the direct reaction without cleanup in a zymo kit. It worked every time.



-stippled-

QUOTE
I used to treat 10ug of DNA in 500uL of reaction with 10x SAM (10 times the NEB reccomendation). I'd do 5 bisulfite treatments from the direct reaction without cleanup in a zymo kit. It worked every time.


These are nearly the same reaction conditions as I have performed my methylation. But how do you test whether the reaction was brought to completeness? Can you recommend a sensitive assay? I'm worried if MSP is sensitive enough to detect traces of unmethylated DNA? All real time PCR assays I found are designed to detect only methylated DNA...

Thanks...

MoB

-MoB-

I do a methylation specific PCR whereby you digest some of your DNA with HpaII and MspI and have normal qPCR primers to a CpG island that you know is unmethylated and therefore methylated after SssI and have this run side by side to the same DNA before treatment. Your HpaII sample should have the same Ct (or same intensity on a gel) as your starting sample.

N

-methylnick-