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production of antisense oligonucleotide by PCR ?

Carrie carrie at fuzzyduck.freeserve.co.uk
Sun Jan 23 17:14:46 EST 2000


R. John Lye wrote in message <38876BC1.79DF445 at virginia.edu>...
>rogier wrote:
>> the tricky part is separating the strands, then isolating the strand
>> you need.
>
>Not really - not if you do a linear amplification using a
>single primer.  I don't have any idea how well it would
>work as an antisense oligo, but you'd get only one strand.


I usually simultaneously prepare both sense and antisense riboprobes using a
linearized dual-promotor vector (T3 and T7) containing my amplified cDNA, as
rogier described. The sense riboprobe acts as a good negative control - I
use them for ISH - as it can't bind to mRNA, which is sense, and it is
possible to verify which riboprobe is which by sequencing if it not obvious
from your experiment. If you linearize a large amount of template, once you
have determined which template produces the antisense riboprobe you can just
use that template stock for producing large amounts of antisense probe.

Carrie







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