the problem on plasmid ligation and transformation

Bill_A_Nussbaumer at Bill_A_Nussbaumer at
Tue Aug 17 08:28:54 EST 1999

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Bill A Nussbaumer at BDX
08/17/99 09:21 AM


I have a couple of questions.

1.  What is a concatemer?

2.  Is it possible to order PCR primers already 5' phosphorylated and hence not
need the T4 PNK step?



> As to your problem
> of cloning I suggest ligating the PCR products to a concatemer to avoid
> problems associated with RE digests close to DNA ends


> Run both samples on an agarose gel. A ladder of
> concatemers should be evident in sample 1, and only momomeric fragments
> again in sample


T4 polynucleotide kinase (1unit; needed to phosphorylate the 5
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=B4 ends of the
product) and ligase (1unit), incubate for 4h at RT.



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