DNA ligation help

[David Micklem]

In article <31B43D9D.2E3C at ukcc.uky.edu>, kostya levay
<klevay at ukcc.uky.edu> wrote:

>Here are my 2 cents:
>
>Once I had very bad experience in cloning 12kb insert into Bluescript 
>with transformation into DH5a. Actually, all of purified plasmids 
>contained deletion of different size. Then I realised that the 
>products of leaking promotors? could be harmful for bacteria and lead 
>me into troubles. Interestingly, that DH5a doesn't have "lacIq" in its 
>genotype, i.e. overproduction of lac repressor protein, inhibiting 
>transcription from the lac promoter. Many other cells do have lacIq.
>I transformed my ligation into JM101, got my construct from the first 
>hit and since then DH5a is on my black list (when I have tricky 
>situations). 
>

Another trick for cloning large constructs is to use a low copy number
plasmid.  It may be that in some cases the deletion problem is not due to
toxic leaking promotors but to the load on the bacteria of producing
_huge_ quantities of a large plasmid.  A plasmid that is maintained at
only a few copies per cell is tolerated better - although its more of a
hassle to get LOTS of DNA for subsequent steps.  (But then how much do you
actually need...)

Good luck,

David

_____________________________________________________________
D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
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Cambridge CB2 1QR             Fruit flies like a banana.
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Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.uk
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