the problem on plasmid ligation and transformation

[Bill_A_Nussbaumer at ms.bd.com]

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

Hi,

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?

TIA,

Bill




> 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

<snip>

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

<snip>

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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