Synthetic Gene

brett 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
>> bases.
>> 
>
>Gilles,
>
>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.
>
>Rafa
                                                 

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


Brett Lindenbach
    
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




More information about the Methods mailing list