How to extract a 20kb vector after double digestion - (Feb/23/2006 )
HI; I have a 20kb vector to be extracted after its double digestion. How can I get more product from the gel or what special kit do I need?
Whether a high melting temperature of the gel will cause any trouble?
THANKS A LOT
what is the size of the fragment you've extracted?...
A 0.5agarose may be slight difficult but why not.
If not, i'm thinking of acrylamide gel (but can't tell the percentage, gradient is optional) and electroelution which works fine...
have you ever tried anything like this?
I have had good luck with their kit for smaller fragments, and they are very easy to use.
After discussion with colleaques, they said it's feasible. They did 17kb with success...
Qiaex II may result in less recovery than for small fragments.
Avoid vortexing and flick gently. you gonna obtain 60%recovery with good purity. THey told me to do a 0.3%agarose gel. For that, for manupilate your gel, saran is strongly recommended to avoid your gel moving... and avoid too to put scotch in comb to make a bigger well : it fraglilizes the gel too much and consequence may be a well destruction when putting out the comb.
Qiaex II may result in less recovery than for small fragments.
Avoid vortexing and flick gently. you gonna obtain 60%recovery with good purity. THey told me to do a 0.3%agarose gel. For that, for manupilate your gel, saran is strongly recommended to avoid your gel moving... and avoid too to put scotch in comb to make a bigger well : it fraglilizes the gel too much and consequence may be a well destruction when putting out the comb.
put in in the fridge to freeze not RT....but i think PA 10-15% will do better,....never tried them for this purpose though...
