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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