Scaling-up of partial digestion

waikhay via methods%40net.bio.net (by waikhay from gmail.com)
Mon Aug 13 15:28:15 EST 2007


Hi all,

I have a question regarding the scaling-up of partial digestions. In my
pilot experiments (serial dilutions of enzyme concentration), I have
determined that the conditions which give my desired fragments are:
1 ug         genomic DNA
0.0125 U  BfuCI (from NEB)
1X           BSA
in 20 ul    total reaction volume
at 37C for 10 minutes

However, I am having doubts on scaling up my digestion. What is the best way
to scale up a partial that would give u an end result that is same as or
very similar to the pilot digestion? Would appreciate if someone here could
help me out, as I have a limited amount of DNA.

On another note, I have tried diluting the enzyme using 1X dilution buffer
prior to partial digestion and it seems that the enzyme has somewhat lost
its activity (as observed from a longer time period used to digest the same
amount of DNA). NEB has warned against this on their website, but may I know
what is the reason a restriction enzyme would lose its activity when being
diluted in its 1X reaction buffer instead of its corresponding diluent
buffer? Would the enzyme lose activity if I were to dilute it using the
diluent buffer to below 1U/ul?

Thanks for the help! :)
waikhay


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