How to clone biotinylated DNA fragment?

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Thu Jul 27 14:50:40 EST 1995


Here's a cloning problem for all cloning gurus out in netland:

A 43 bp oligo was designed as completely self-complementary. To be used
for a gel-shift experiment, it was 5' biotinylated. The rub...when heated
to 90 degrees C and slow cooled, alot of secondary structures are seen
on a gel -> not a good probe for a gel shift. Several ideas were to:

1. Change the annealing temp. or conditions....Hmmmm...to what?
2. S1 nuclease the ssDNA cruciforms and leave the ds annealed DNA...
   but the nuclease chewed all structures to bits.
3. Clone the beast and use plasmid DNA for the shift...question:
   If 5' biotinylated, can you blunt end clone the DNA? OR is it better to
   poly-A the 3' ends (ala Taq polymerase) and T-vector clone it? Would
   this work?

Is there a better way to get only annealed dsDNA without buying a new oligo?

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
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