DNA ligation help

John Ladasky ladasky at leland.Stanford.EDU
Mon Jun 3 12:47:29 EST 1996

	Followups have been limited to bionet.molbio.methds-reagnts.

In article <4opneu$nke at newz.oit.unc.edu>, chunlin xin <xcl at med.unc.edu> wrote:
>Hi, there, I have trouble in DNA ligation, the case below:
>          Insert Fragment: 16.3kb SalI Frag.
>                   Vector: Bscrip-KS(-) SalI cut plasmid or other 
>                           salI cut expression vector
>          It looks like no ligation problem, but in fact, I cannot 
>success to ligate them and transfer them into DH5a no matter I use 
>chemical or electroparation method, Have anybody  ever meet the same problem?
>How can I solve them? Any suggestion is appreciated! Thanks for you attention!

	A 16.3 Kb insertion is quite large.  DH5-alpha, and most other
normal strains of E. coli, cannot reliably maintain plasmids larger than
10 Kb.  (I'm not sure why this is so.)  So your ligation and transformation
may both be successful, but then you are not getting any ampicillin-resistant
colonies.  Can you subclone this fragment?
Unique ID : Ladasky, John Joseph Jr.
Title     : BA Biochemistry, U.C. Berkeley, 1989  (Ph.D. perhaps 1998???)
Location  : Stanford University, Dept. of Structural Biology, Fairchild D-105
Keywords  : immunology, music, running, Green

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