oligo cloning

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Mon Feb 7 17:26:17 EST 2000


In article <389F4115.878E178A at pombe.usask.ca>, Colin Rasmussen <
colin at pombe.usask.ca> wrote:
>
>
>Caroline Szymeczek-Seay wrote:
>
>> I'm cloning (or trying to clone) a double stranded blunt
ended oligo (a
>> 47mer) into pGL3 enhancer vector from Promega.  Vector is cut
with SmaI
>> and CIPed.  Oligos are kinased.
>>
>> My question:  is there a rule of thumb for amount of oligos
to add to
>> vector?  I'm trying a 10-fold molar excess, a 5-fold molar
excess and an
>> equimolar amt of oligo relative to vector. I'm getting no
positives.
>> This is a little hairy also because there's no blue/white
screening for
>> this vector.
>
>What I would do is to use unkinased oligos, and leave the
phosphate on the
>vector.  Then ligate with diminishing amounts of vector while
keeping the
>insert concentration the same.  Do a plating onto X-gal plates
and pick
>white colonies.  . . .

Um, she just said her vector isn't blue/white "screen-able."
Anyway, I've found sometimes that small inserts still give blue
colonies (heck, I once had a a 1 kb insert give blue colonies --
that had me going for awhile).

This suggestion may be too late for the original poster, but
where
possible, I prefer to use "adapters" by engineering sticky ends
on
the oligos that will ligate in a unique way into a doubly cut
vector, and also to put a unique restriction site in the oligo,
if
practical.



Nick Theodorakis

nicholas_theodorakis at urmc.rochester.edu

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