Re: gene synthesis
Anthony Yeung (AT_Yeung@FCCC.edu)
Mon, 21 Jun 1999 10:00:12 -0400
Shalom:
1.1kbp is what we did,in collaboration with my client Dr. Jaffe, so that
is no sweat either, just four hours of work to plan the experiment and two
weeks to execute. All our designer gene have worked, and gave little dump
trucks of foreign proteins in coli, soluble and active too. All E. coli
codon usage!!! Now you need to pay attention to primer and template design.
I used 3' overlapping templates suitable for primer extension. I use about
25-35 bp overlaps with the nucleotide in one template next to the 3' of the
other template as a T so that the A gets inserted ASAP to increase the
operlap by two nucleotides. The 180mers are double deblocked to be sure.
Gel purification may give no band, so cut at the blank where the band is
supposed to be. I have a true pro here if you should need assistance.
Electroelute the DNA. Plan 8 PCR primers so they do not primer each other.
Balance all Tm. First do reaction with AmpliTag to make sure you are likely
to succeed, then switch to PFU and work out the conditions. Clone each
amplification stage if the oligos are not purified, and select the correct
products for further PCR and assembly. Mutation occurs only in the first
PCR off the synthetic oligos. It appears as though the synthesizer is able
to pause several bases and then continue the synthesis, kind of like the
I have to go, I am particularly loaded these two months. Please email me
in future as you need precise conditions.
Hope this helps!
Tony
At 01:48 PM 6/20/99 -0400, Shalom David Goldberg wrote:
>
>The gene is actually around 1100 bp long - I mistyped - this is the
>difficulty.
>
>Shalom Goldberg
>
>---------- Forwarded message ----------
>Date: Sun, 20 Jun 1999 00:04:41 -0400 (EDT)
>From: Shalom David Goldberg <sdg11@columbia.edu>
>To: abrf@aecom.yu.edu
>Subject: gene synthesis
>
>
>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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