Pulse Field Gel Electrophoresis (PFGE) - Secondary Restriction Enzyme Problem (Oct/16/2008 )
Any suggestions on what could be the cause of the secondary restriction enzyme not working properly and/or any suggestions on improving how the secondary restriction enzyme works?
Thank You!
do you use a control (e.g. cutting with the second RE only and running PFGE) ? how do you know it is not working ?
RE work all the time, no matter what, I've been using enzymes that had been sitting in the freezer for 11 years and they'd still work as in their early days, it is however also true that some enzymes are really sensitive to freeze-thaw and will not work after their expriy date or less if misshandled.
If the first RE is working, I see no reason why your template wouldn't work for the second enzyme (given that there are RE sites 
)
- do you remove the first enzyme+buffer ?
- if not, is the first buffer compatible with the second enzyme ?
- do you use the correct reaction temperature?
- do you use enough enzyme?
RE work all the time, no matter what, I've been using enzymes that had been sitting in the freezer for 11 years and they'd still work as in their early days, it is however also true that some enzymes are really sensitive to freeze-thaw and will not work after their expriy date or less if misshandled.
If the first RE is working, I see no reason why your template wouldn't work for the second enzyme (given that there are RE sites
)- do you remove the first enzyme+buffer ?
- if not, is the first buffer compatible with the second enzyme ?
- do you use the correct reaction temperature?
- do you use enough enzyme?
Dude what restriction enzyme is that?? 11 years .. I 'm impressed!