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bisulphite treatment by kit - need information about it and successfull in DNA treatment (Nov/09/2006 )

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hi , I am a new member , and I would information about bisulphite treatment of DNA,I use EZ DNA Methylation Kit by Zymo RESEARCH.
I extract genomic DNA, treat it by KIT in question, make a PCR and then TOPO-TA cloning and sequence 10 colonies...
but I have only few C converted in T and I worrry about the treatment doesn't function very well, but how can I understand if it works or not?

-full-methy-

Hi. I think you may need a control for the bisulfite treatment. Extract DNA and treat it SssI methyltransferase (NEB). This enzyme will add methyl group to C in 5'-CG-3' sequence. SssI treated of bisulfite modified and unmodified DNA is used as substrate in a PCR reaction. The differences seen in sequencing PCR products indicate methylated verses unmethylated regions.
Hope this will help.

-mrpcr-

QUOTE (mrpcr @ Nov 10 2006, 03:59 AM)
Hi. I think you may need a control for the bisulfite treatment. Extract DNA and treat it SssI methyltransferase (NEB). This enzyme will add methyl group to C in 5'-CG-3' sequence. SssI treated of bisulfite modified and unmodified DNA is used as substrate in a PCR reaction. The differences seen in sequencing PCR products indicate methylated verses unmethylated regions.
Hope this will help.



thx, do you have a protocol of reaction for SssI (New England)?
must I use genomic DNA or can I use a PCR product about 700bp?
but Do you have used this EZ methylation kit?

-full-methy-

QUOTE (mrpcr @ Nov 10 2006, 03:59 AM)
Hi. I think you may need a control for the bisulfite treatment. Extract DNA and treat it SssI methyltransferase (NEB). This enzyme will add methyl group to C in 5'-CG-3' sequence. SssI treated of bisulfite modified and unmodified DNA is used as substrate in a PCR reaction. The differences seen in sequencing PCR products indicate methylated verses unmethylated regions.
Hope this will help.




sorry I think that I need a DNA that is not methylated and i can test if my kit functions.
in the DNA that I sequenced I have C not methylated near T and A. do you know this situation?

-full-methy-

Hi Full-Methy,

as only C's in CpG's should be methylated (at least in mammalian cells), you have a quality control for your bisulfite reaction when looking at non-CpG C's. All of them should be transformed to T's. If, for example, you have a sequence like this:

5'-ATCTCTCATTCTCACGAT-3'

it should look like this after treatment, assuming the 3' C in the CpG was methylated:

5'-ATTTTTTATTTTTACGAT-3'

If you have any other Cs left, the modification did not work well!
It can also help to design primers that include some non-CpG-Cs converted to T. PCR should than only amplify the sufficiently modified DNA.

But fully methylated DNA controls should help too and can be used to calibrate your measurements.

The protocol for SssI treatment is provided by NEB: http://www.neb.com/nebecomm/products/protocol96.asp

If your Modification Kit is refusing to work properly, try the Epitect Kit from Qiagen - that works fine in my hands!

Good luck,
Kr├╝mel

-kr├╝melmonster-

Before you go and start SssI methylase experiements can I suggest you have a look at your primer design???

inefficient primer design can lead to misamplification of unconverted templates, you need to design your primers such that they target fully converted DNA..

post your primers for all to see and I bet you, your primer design is not optimal.

The zymo kit works very well in my hands but like with the home brew method, you loose much of your starting gDNA to degradation. I put my two cents in for methyleasy, it's great if you have very little starting dna to start with.

Methyleasy Kit

-methylnick-

QUOTE (methylnick @ Nov 12 2006, 10:49 AM)
Before you go and start SssI methylase experiements can I suggest you have a look at your primer design???

inefficient primer design can lead to misamplification of unconverted templates, you need to design your primers such that they target fully converted DNA..

post your primers for all to see and I bet you, your primer design is not optimal.

The zymo kit works very well in my hands but like with the home brew method, you loose much of your starting gDNA to degradation. I put my two cents in for methyleasy, it's great if you have very little starting dna to start with.

Methyleasy Kit



Hi , I amplified, my gDNA treatment with bisulfite, using primers that I are not modified for AZA and bisulfite amplification...and I think that my kit is not working well. is it right?

-full-methy-

full-methyl,

I am afraid you maybe incorrect, could you post your sequence of intererst and the primers you used for amplification?

-methylnick-

QUOTE (methylnick @ Nov 13 2006, 01:42 PM)
full-methyl,

I am afraid you maybe incorrect, could you post your sequence of intererst and the primers you used for amplification?



I think that EZ Kit doesn't function in my hands ....maybe am I using too much DNA (500ng for treatment)?
sorry can you indicate as I can draw my primers? what do you use for PCR amplification ? Hot StarTaq(qiagen)?

-full-methy-

QUOTE (full-methy @ Nov 15 2006, 11:59 AM)
QUOTE (methylnick @ Nov 13 2006, 01:42 PM)

full-methyl,

I am afraid you maybe incorrect, could you post your sequence of intererst and the primers you used for amplification?



I think that EZ Kit doesn't function in my hands ....maybe am I using too much DNA (500ng for treatment)?
sorry can you indicate as I can draw my primers? what do you use for PCR amplification ? Hot StarTaq(qiagen)?


I've loaded over 1ug into the EZ kit, and i get excellent conversion.

There's a new kit that claims to not deplete as much DNA. MethylEasy from HGS.
I have a test kit here, but i don't have the time to play around with it yet.

Any opinions on the MethylEasy kit anyone?

-sneth-

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