brett at BORCIM.WUSTL.EDU
Mon Sep 9 20:27:01 EST 1996
>On Fri, 6 Sep 1996, Gilles Truan wrote:
>> It looks like the formatting disappeared in the posting. Anyway, I can
>> expalin it with words. The total gene is around 1400 bp. We have made
>> synthesize 50 oligos, 48 that are 60-mers, 2 that are 20-mers. The
>> strand + begins with 24 60-mers and end with 1 20-mers, the - strand is
>> exactly the same, in the opposite direction. In other words each oligos
>> hybridize two complementary oligos, one with 20 bases, the other with 40
>You have really few chances to get the ligation right. I have never tried
>so many fragments (a four fragments ligation is already a pain) but I
>will tell you what I did:
>- Mix and heat each pair of oligo, cool slowly.
>- Mix all the previous mixes, heat at 55C (no more than the Tm of your
>pairs), cool slowly and ligate overnight at room temperature.
>- The amount of final product is too low for direct cloning, so I did a
>PCR with oligos matching the ends of the final ligation product, so *with
>luck* you will get something clonable. You can gel purify and digest with
>enzymes which sites are included in your oligos, or use TA cloning.
>Alternatively, you can make much longer oligos (ask for the maximun size at
>your oligo provider; we can make 120 bp oligos without much trouble, so
>shop around), and in this way your ligation product will be more abundant
>for the PCR. Knowing the power of amplification of PCR, you should get it.
>You could make it also in several steps and ligate them later.
>Good luck, you will need it.
I disagree. This construction should be no problem. Perhaps Rafael doesn't
comprehend the amount of overlap your oligos will have. T4 Ligase will have
a field day once you get all these guys annealed. Good luck
Program in Immunology
Washington University - St Louis
brett at borcim.wustl.edu
"I own my own pet virus. I get to pet and name her." - Cobain
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