Gentle Plasmid prep protocol needed
peter.cherepanov at uz.kuleuven.ac.be
Tue Jul 14 10:08:00 EST 1998
Years ago we did nested deletions using Quagen-prep DNA and
ExoIII/MungBean method. No CsCl gradients.
I didnt notice any problems there (the insert lengths to sequence were
1.5 kb - for much longer pieces it is not very sutable).
It is important to purify the band of shortened DNA by gel anyway - if
you had a nick in the middle somewere, then the DNA would be cut in the
middle, so you will get rid of it cutting the right band from gel. Then
you have 0.1-5 ng of DNA which is very easy to religate.
Although it might be that you are using a low copy vector or a large
plasmid (?) then the quality of the prep may be too low indeed (We
sequenced two overlapping clones instead of the large one).
By the way, ExoIII is my favorite enzyme. Use some excess of it, and at
37C. It gave more reproducible deletion rates, in my hands.
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