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Double Digestion Restriction Enzyme - Troubles with double digestion (Jan/07/2009 )

Hi Everyone,
I gotta a question for all of you , i will really appreciate if you can help me
- I gotta a plasmid (pCoiZA 8.1kb) i wanna cut it into sites PstI and XbaI
- My PROBLEM::::: have to do double digestion but still cannot get it , i did how it says in the procedure but still doesn't function

- I'm using RE from Takara company, they said to use 1X Buffer M but doesn't seem to function, then i read in one paper that i should use RNase 1mg/ml but doesn't function.

Please really need it to get it fast and cannot do 2 simple digestion, coz I always loose DNA when i have to do the clean up for next RE, so really don't know all ur suggestion are welcome.

Please reply ASAP


What is the efficiency of PstI in Buffer M? Takara recommends using Buffer H for PstI. If you aren't using a buffer that results in high enough efficiency that would explain your problem. Also for problematic digests, I usually digest overnight at 37 deg C.


I see Takara say you should have 100% efficiency with buffer M.
What result do you get? No digestion at all? It seems strange that neither enzyme would cut.
How long do you allow the reaction to go on?


[code][/code]I use 2ul each enzyme,incubate overnight and next day there is no band is it all degradeated ???