Tue Jun 11 00:15:17 EST 1996
Here are the easy suggestions: Gel purify your vector since unlinearized can be
present but not visible on the gel. Dephosphorylate the vector (the new
heat-labile phosphatases work nicely) to reduce your background. Increase the
concentration of your vector (i.e. up to 100ng) and insert too. Don't use
blue/white selection since if the insert is toxic you may be killing off what
you want. Finally, try a low copy plasmid. All these are typical suggestions.
Other ideas, why do you CsCl band your DNA? Having worked with chloroplast DNA,
I know that as long as you purify the organelle away from the nucleus, one
should be able to get clean DNA. This would avoid losses incurred in the
banding process as well as those harmful nicks when pulling the band under UV.
In addition, if you have banded your DNA 3x, there shouldn't be any RNA present
and thus RNAse treatment is superfluous. Also, why do you not gel purify your
fragment? If there is too little DNA to visualize, isolate from the gel the
correct molecular weight region, make sure to add the digestion buffer to your
molecular weight markers or you will be off.
hope these ideas help,
amief at candelab.berkeley.edu
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