Subcloning: Loosing my favorite gene

ChenHA via methods%40net.bio.net (by hzhen from freeuk.com)
Tue Feb 13 20:44:59 EST 2007


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.  

If you have sequenced it, then you might be able to check if there is
deletion and where the deletion occur.  I think GFP is smaller than
800bp, so you got some other sequence attached to the GFP, check if
there are sequences which is same as sequences in the vector as
repeated sequence can cause deletion.



>Also, I intend to put my GFP into this SgrAI cut site.
>
>

My preference for cloning is to PCR up the GFP fragment with 2
restrictions site, then clone into your gene containing the same 2
restriction sites (i.e. directional cloning).  Of course, your problem
could be that you can't find two suitable restriction sites.  I just
hate cloning into single site and I would rather do a mutagenesis step
first to introduce suitable restrictions sites before cloning, but I
understand some people would find the extra steps unncessary.


>
>> >
>> What do you mean by a linearized gene?  Genes aren't naturally circular.  I
>> presume you mean that you've cut the gene with SgrAI.
>>
>
>
>> You should ligate the GFP to your gene before cloning it into the plasmid.
>
>The idea behind this project is to clone a flourescent tag inside the
>gene.  The gene is cleaved at both the N and C termini so I can put
>the gene downstream of GFP.  My source of GFP is clone I built from a
>protein expression vector.  My pGFP is being cut with SgrAI,
>electrophoresed and gel purfied.
>
>If you're able to think of anything else I'd appreciate hearing it.
>

If you have GFP and you can express it in your vector, then one the
easiest way is to transform your ligation mixture, then transfer the
colonies  you get into another plate with IPTG and you should see the
colonies lighting up under UV when you have a successful clone.   To
transfer the colonies you can either first plate on a membrane placed
on top of agar, then transfer the membrane with colonies growing on
it, or you can use a velver pad of some sort to do the transfer.

That strategy, of course, depends on whether you get soluble GFP
protein that folds properly for it to work inside your gene.



>-A
>
>



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