subcloning large fragments

Bernard Murray bernard at elsie.nci.nih.gov
Tue Feb 4 23:28:02 EST 1997


In article <pmiguel-0402971847050001 at opie.bio.purdue.edu>, 
pmiguel at bilbo.bio.purdue.edu says...
>
>In article <199702042236.QAA20303 at borcim.wustl.edu>,
>brett at BORCIM.WUSTL.EDU (brett) 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.

>Strange.  I've never noticed anything recombining in DH5a.

I also vote for DH5alpha as a dependable general purpose strain.
I've never had to make a construct as heroically large as these
two posts indicate but I would lean towards DH5 derivatives as hosts
because of the deoR mutation in these strains (and DH10) that
(at least according to the Gibco/BRL catalogue) "allows the uptake
of large plasmids".
	(Interestingly, Invitrogen's INValphaF' strain which is
usually considered a relabelled version of DH5alphaF' is not
listed as deoR so this may be worse for large plasmids).

Can I also pick the brains of creators of large plasmids...?
Do you think you can play with the transformation conditions
to allow selective uptake of larger plasmids (maybe longer or
shorter duration of heat shock) or do optimised conditions
obtain to all sizes?  If smaller (vector alone) plasmids are
taken up *much* more easily then this would argue for the use
of something like the ccdB gene selection to boost the chances
of obtaining the desired recombinant after ligation.
	Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)




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