MBO1 digestion - (May/28/2009 )

Hello,
I am trying to do expt on ALU by digesting bisulfite modified sample with MBO1. So did BS modification, PCR, got single bright band at 150 bp. Then 1 -2 microgm of PCR product was kept with MBO1 at 37 degree for 3/ 4 hrs, tried overnight also. It should give me digested bands 110 and 40 bp.
But i am getting same band only at 150 bp after digestion also, no other product.
Infact in gel when i ran digested samples next to undigested one, the digested band appears to be a little bit higher than 150 bp.
Where's the problem. I dont have any idea why digestion is not working. Any other suggestions?
Any help will be appreciated.
Thanks,

maybe MBO1 restriction site is not in your amplicon.

As for the amplicon seemingly "larger" I would say it would be because of the salt/reaction buffers making the migration of the amplicon run differently, I would assure you that if you mix your undigested amplicon with the reaction buffer of your digestion but without the enzyme, they will then run the same.

nick.

methylnick on May 28 2009, 03:11 PM said:

Thanks Nick for your reply. The thing is, I am doing COBRA assay for ALU. and there are atleast 2-3 papers where they adapted same technique for ALU. I am just trying to replicate that in my sample. I am sure, my amplicon have the MBO1 Restriction site.
I guess, you are right, it might be buffer problem. I was using promega enzyme with the buffer they supplied.
In one paper they used NEB, so let me try with NEB buffer (which is a little bit different than promega).
thanks again,

Changing buffer did not work.
Mbo1 cuts only methylated site. So only accptable explation, that site is not methylated in my samples, will try with methylated samples.
Thanks,

This is just to update my problem i mentioned before.

I ran Native Page (12%) as my DNA fragments were 125 and 152 bp.
i could see clearly the two bands in the digested sample and only 152 band in undigested one.
Thanks,