method of using the CpG methylase enzyme - (Oct/12/2005 )
i am in the process of standardising my bisulfite method for studyin the methylation status of certain tumor suppressor genes, i have therefore ordered a CpG methylase enzyme which would act as a positive control. i have also received a buffer alongwith the enzyme. but i dont have any kinda literature/protocol which suggests about the amount of enzyme and buffer as well as conditions i shuld take care of in order to methylate my DNA with the enzyme and the quantity of buffer is should use.please do help me i am kinda stuck here.thanks n hopin i really really get a way out of this .....
Hi researcher 81,
I take it you have some SssI methylase at your disposal? the enzyme should come with a product sheet and this will tell you the amounts of DNA and enzyme required. The NEB site will have the sheet for download on their site also.
You can also check to see if your DNA is methylated by a simple HpaII and MspI digest where if your DNA was methylated the HpaII digest would reveal no digestion, where in MspI you will see digestion.
Here is what I did.
50ul reaction=5ug DNA+1ul SAM (32mM) + 1ul SssI (20,000u/ml) +5ul 10XNEB Buffer +H2O
Set up the reaction at 37C for 4hrs. Then clean up the product using the Zymo kit.
SAM and SssI are ordered from NEB.
hey thanks a tonn i am really very very grateful that i have found a way out. i will try out the protocol you have suggested n GOD BLESS YOU!!!
Does this method of using SssI CpG methylase work well with fairly intact genomic DNA? not just plasmids?
I tried using the 4 hr reaction, but it doesn't seem as though I have achieved full methylation of all CpG sites.
You won't get fully methylated of all CG sites probably. Somebody told me to do the reaction twice or maybe three times.
Could I just do a longer incubation with the CpG enzyme? say overnight rather than simply a 4 hr? Or is there an issue with the enzyme losing activity over time?
methylation requires SAM,
this may have depleted if you are tryiing to methylate the entire genome. I am not too sure why you would want to do this, but it would be prudent to just complete 2 or 3 repeats of the reaction.