Subcloning: Loosing my favorite gene

Jose de las Heras via methods%40net.bio.net (by josenet from tiscali.co.uk)
Wed Feb 14 18:32:10 EST 2007


"ChenHA" <hzhen from freeuk.com> wrote in message 
news:kjo4t25j2dbt2935icr0ovrtd3jmr67bbe from 4ax.com...
> On 13 Feb 2007 15:56:12 -0800, "Allan" <allan.debono from gmail.com> wrote:
>
>>Hi Peter,
>>
>>Thanks for your input.  I don't think I have explained myself well
>>enough.
>>
>>> This cut site is in the gene insert, not the vector.
>>
>>My gene at this point is in pUC19 yielding pMyfavoritegene (pMFG).  I
>>cut it with SgrAI to linearize and dephosphorylate and gel purify only
>>the resultant 6.3 KB band.  At this point I don't know how a
>>supercoiled pUC19 can migrate at 6.3 KB with the linearize pMFG.
>
> Uncut plasmid can run with multiple bands, so it is entirely possible,
> though I think unlikely in your case, to get pUC running at higher
> apparent size.

indeed!

in fact, if you gel-purify a larger band, and run it on a gel again, you may 
be able to even see some of the lower size bands (if you have enough 
material, of course!). There will not be a lot of it, but you don't need 
much to successfully transform and obtain lots of clones.

It's just the way electrophoresis works... at the bottom of teh gel you will 
only get the small stuff, but at the top you will get *mostly* large stuff, 
but also a small % of "renegade" small stuff...

Jose 




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