seperating 3.5 and 3.3 kb in agarosegel

Michael Sullivan via methods%40net.bio.net (by mlsulliv from wisc.edu)
Thu Jan 21 09:51:55 EST 2010


As Peter suggested, if this is for recloning into another vector, it  
may be easier to "shotgun" clone both fragments and sort them out  
later since it is relatively easy to distinguish small differences in  
fragment size, but sometimes difficult to cleanly isolate such  
fragments.

If you plan to clone the fragment into a vector that has a different  
selectable marker than the original clone, you can also use the  
marker to sort our events. An example: I often build gene silencing  
constructs in an amp resistant vector, but the silencing cassette  
ultimately needs to be inserted into a plant transformation vector  
(which usually has kan or spec resistance). For this cloning, I  
usually digest the original amp construct and shotgun clone both  
fragments into the plant transformation vector. For the colonies that  
come up (i.e. are resistant to whatever drug is appropriate for the  
plant transformation vector), I pick each onto two plates, one with  
amp and one with the other drug. The clones that cannot grow on amp  
(i.e. they lack the vector sequence from the original clone) are the  
ones you want.

Hope this helps.

Mike

On Jan 20, 2010, at 4:02 AM, Senthil Thyagarajan wrote:

> Dear all,
>
> I need to seperate DNA bands of size 3582bp and 3324bp and elute  
> the former
> one. Can anyone suggest me the percentage of agarose gel, that  
> could help me
> seperating these bands. Btw, there are no good restriction enzyme  
> sites that
> break up the backbone and am left with no way other than seperating  
> these
> two bands on the gel.
>
> Your help is very much appreciated. Please write to me asap. Thanks  
> a lot in
> advance.
>
> Regards,
> Senthil
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---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)



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