How Much Ligase Do I Need?

Aawara Chowdhury aawara at FEMA-trailer.org
Sat Aug 19 17:34:26 EST 2006


In <1156022947.729908.158790 at i42g2000cwa.googlegroups.com>,
 htert2020 at yahoo.com <htert2020 at yahoo.com> wrote:

> Thanks for your response.  The concentration of DNA will not give me
> any undesirable concatemers because the ratio between the two different
> oligonucleotides will be, more or less, one to one.  Moreover, an oligo
> will have a very difficult time forming concatemers because the only
> way to join two oligos in that manner is through blunt end ligation...
> there are no "sticky ends" because an oligo is one-sided.  Here is a
> text diagram to illustrate.  For best viewing, copy and paste it into
> Windows Notepad or other word processor that displays text in fixed
> character width font:
>
>        OOOOO
>       O     O
>      O       O
>      O       O
>      O       O
>       O  .  O
>        OOOOO        <== 36mer oligo circularizes where the "." is
>  TTTTTTTTTTTTTTTTT  <== template 18mer oligo
>                     The "." is where the ligase acts


Andrew -

I'm a little curious - is your 36-mer entirely single-stranded; so when
you add the 18-mer, it looks like a nicked circle?

If its double-stranded, I don't know if your strategy will work for two
reasons - a) there's plenty of evidence that DNA phasing plays a large
role in the proper juxtaposition of ends for molecules under 200 bp,
and b) molecules under about 70 bp will not circularize (without assistance
from a DNA-bending protein like HMG-1 or HMG-I/Y).

AC

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