I have done three gene synthesis of 1.1 kbp each using recursive PCR using
PCR primers to join templates of about 180 bp long. At these lengths, oligo
purification is a must to lower the frequency of deletions and mutations
from over 50% of the products to close to 10%. . We use PFU and sequence
each 340bp product before further assembly. 100bp is a piece of cake. Just
gel purify two sixty mers and join them by primer extension.
Good luck to you!
Tony
At 12:04 AM 6/20/99 -0400, Shalom David Goldberg wrote:
>
>Hi All,
>
>I need to synthesize a gene that is about 100 bp long. I'm planning to
>use a PCR-based method, using overlapping oligos. I would really like to
>know if there's anyone who has done a PCR-based gene synthesis, especially
>of a long gene, how well it worked, and suggestions for construction of
>the oligos (ie length of overlap, oligo size, etc.)
>
>Thanks,
>Shalom Goldberg
>
************************************
Dr. Anthony T. Yeung, Ph.D.
Director, Fannie E. Rippel Biotechnology Facility
Member, Institute for Cancer Research
Fox Chase Cancer Center
7701 Burholme Ave. Philadelphia, PA 19111
Voice: 215-728-2488
FAX: 215-728-3647
email: AT_Yeung@FCCC.edu
http://www.fccc.edu/research/labs/yeung/
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