Re: [DIYbio] Molecular Inversion Probes Debugging

On Thu, Mar 12, 2015 at 2:06 AM, Mega [Andreas Stuermer]
<masterstorm123@gmail.com> wrote:
> Hi guys!
>
> For the lab exercise we designed molecular inversion probes. The first batch
> we designed didn't work, so we ordered a probe from the original paper and
> it still didn't work...
> The main mechanism is the probe is circularized if it fits to the template.
>
> The single stranded probe is 115 bases long. How can we check if
> circularization worked without radioactive labeling or expensive stuff?
>
> I was thinking of running a gel - circular 115 bases should run slower than
> linear 115 bases. Though the concentration is very very low - attomols - so
> no chance to see on a gel.

Maybe evaporate some buffer to increase concentration, then introduce
to HPLC or capillary electrophoresis. If you have an old DNA sequence
available (sanger) you could probably use that for running your
sample, as long as the transfer losses aren't too great (from sample
vial, sucked into syringe/needle, then into the column).

> Also, just using one primer and making multiple tandem copies (to get 230,
> 345, 460, ... bases) and subsequent amplification won't work (Polymerase
> won't kick away the primer)...

Not sure what you mean, why would the polymerase prevent melting of the primer?


If the DNA was nicked... I wonder if you could ligate on some bigger
sequence that would stick more to a spin-column, and not elute as
quickly on the circular stuff.

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