At 13:20 2/4/97 -0800, Frederic G. Barr Lab wrote:
>When subcloning DNA fragments larger than 5 kb into pBluescript, I
>frequently recover DH5a mini-prep clones with various-sized deletions or
>no inserts. What reagents (bacterial strain, plasmid vector, restriction
>sites) should I be using so that I can subclone large (5-15 kb) DNA
>fragments that are intact?
In my various travels, I've cloned anything from 200 bp upwards to about 14
Kb. I've dropped them into pSP72 and pBluescript without too much
difficulty. The problem with the bigger ligations isn't accomplishing the
ligation but getting the product into a bug. That drops precipitiously as
a function of size. I fiddled with Electroporation and only managed 3 X
10^7 cfu/ug with bluescript (alone), that dropped to 4X10^4 using
BlueBacIII (10.5 Kb) and then to 3X10^3 cfu/ug using an intact cosmid.
Note: these were intact vectors, not products of ligation. Ligations of
larger fragments are subject to the usual "gremlins" of ligation, this
added to the "fall-off" of transformation can make life very hard... - it
took me three separate ligations (with three different preps of vector and
insert) and screening by hybridization for me to get one clone of pSP72
with a 14 Kb insert...
I've managed to get "big" things in using pSP72 and bluescript. For bugs,
I've mainly used SURE cells, both XL-1 and XL-2's, and if I'm trying to get
something big in, TOP10F'. using these, I've not seen
deletions/rearrangments - concatamers of small products, yes.
Hope this helps,
David L. Haviland, Ph.D.
Asst. Prof. Immunology
University of Texas - Houston, H.S.C.
Institute of Molecular Medicine
2121 W. Holcombe Blvd.
Houston, TX 77030
Internet:"dhavilan at imm2.imm.uth.tmc.edu"
Voice: 713.500.2413 FAX: 713.500.2424
" Sometimes you're the windsheild, sometimes you're the bug."