separate DNA fragments of same length

Morris F. Manolson mm6y+ at andrew.cmu.edu
Sun Feb 7 18:04:41 EST 1993


>      Is there anyone knowing how to separate DNA fragments of exactly same
>length (about 150 bp) with different sequence? These fragments were isolated
>by PCR using degeneracy primers. I don't want to screen hundreds of colonies
>by DNA sequencing. Also, I don't want to lose many amplfication products.

Our approach was to subclone the appropriate size PCR fragment
into bluescript. We then did about 24 minipreps from the resulting
transformants, followed by double-standed T-lane sequencing (ie: for each clone
we only sequenced using one ddNTP). By comparing the single
T-lanes from each clone, we saw that the 24 clones were composed
of 6 groups, which we then sequenced completely.  
Of course none of the 6 clones were what we wanted but that
is yet another story.  Morrie 



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