cloning large fragments

Burkhard Hassel Burkhard.hassel at t-online.de
Tue Mar 28 03:36:44 EST 2000


I cloned several Sal I fragments of ~ 15 kb from lambda EMBL phages
into pBLUESCRIPT. But I never got colonies, when the vector was CIPped
(don't know why, maybe because I didn't use electroporation). So I
cloned the DNA from the exciced band into uncipped vector with blue /
white selection. Molar relation of insert to vector was 3:1. The
plates where full of blue colonies with few (<10) white ones.
Once I got a concatemer, so this insert was around 30 kb.
DH5 alpha did better than XL1blue, at least in my hands.
Burki

On Mon, 27 Mar 2000 12:32:37 +0100, "P.J. Shaw" <pjs14 at le.ac.uk>
wrote:

>I routinely subclone inserts from lambda DASH clones. Usually, it's no
>problem to get the whole insert cloned using the SalI sites in the
>polylinker, if there's no internal sites. Don't bother gel-purifying; just
>ligate an excess of cut fragments to bluescript which has been
>dephosphorylated with SAP. Transform by electroporation and pick whites
>(usually not a lot!). DH5a is OK, sometimes SURE cells are needed if you
>get deletions.
>
>Phil.
>
>pjs14 at le.ac.uk  
>





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