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From 2012.igem.org

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Revision as of 23:28, 17 September 2012

Gene Synthesis

1. Generate Oligo's
   1. Go to: http://helixweb.nih.gov/dnaworks/
   2. Set parameters
      1. Enter you job title and email
      2. Choose E.Coli Class II for codon frequency
      3. Set Annealing temperature to 60
      4. Maximize oligo length for cheapest oligo (60 for most companies)
      5. Set Number of solutions = 10
      6. Select Non-degenerate sites to avoid
         1. Bio-Brick requires EcoRI, XbaI, SpeI, and PstI, others can be chosen if desired
      7. Leave rest of options default
   3. Enter Sequences
      1. Click "Add Sequence Field" twice under Sequence formats
      2. Imput your header sequence (select Nucleotide, this contains your cut sites,spacers, etc for subsequent cloning)
         1. eg. GGATAGGA CATATG
      3. Enter you protein sequence (select protein)
      4. Imput your tail sequence (select Nucleotide, this contains your cut sites,spacers, etc for subsequent cloning)
         1. e.g. CTCGAG ATTCGATG
   4. RUN
      1. If nothing is running make sure there are no blank new lines in your sequence section!
   5. Choose your favorite oligo set to synthesize your gene
      1. Usually look for the best scoring with the closest Tm's and oligo lengths
   6. Design two additional oligos to amplify your gene
      1. A FORWARD and REVERSE oligo that complements your final DNA sequence with a Tm of 65. Just copy from the 5’ end of your first and last oligo from oligo’s reported from DNAWorks until you have a calculated Tm of 65 (20‐30bp, +/‐ 1deg). Try to make sure then ends are either G/C.
   7. ORDER
      1. I often try and order in plates (easier if ordering a lot) and make sure that the nmol of oligo is normalized. For IDT this is free, but that may differ for other companies.
2. Synthesize Gene
   1. Dilute all oligos to 100uM
      1. Resuspend oligos with water: uLs of water = concentration of oligos (in nm)*10
   2. Mix together
      1. add 5uL of each into a new master tube
   3. Setup Synthesis PCR Reaction (have tried Taq, Vent, and PfuTurbo. Results are always best with Phusion)
      1. 1uL Oligo Mix
      2. 1uL 25mM dNTP's
      3. 10uL Phusion HF Buffer
      4. 0.5uL Forward Oligo
      5. 0.5uL Reverse Oligo
      6. 0.5uL Phusion polymerase
      7. 36.5uL diH2O
   4. Synthesis PCR Reaction
      1. 98C - 30s
      2. 98C - 10s
      3. 63C - 10s
      4. 72C - 30s/kb target gene
      5. Repeat 2-4 29x
      6. 72C - 5min
      7. 10C - forever
   5. Setup Amplification PCR Reaction
      1. 1uL FROM UNPURIFIED SYNTHESIS REACTION
      2. 1uL 25mM dNTP's
      3. 10uL Phusion HF Buffer
      4. 0.5uL Forward Primer (Tm 65)
      5. 0.5uL Reverse Primer (Tm 65)
      6. 0.5uL Phusion polymerase
      7. 36.5uL diH2O
   6. Amplification PCR Reaction
      1. 98C - 30s
      2. 98C - 10s
      3. 63C - 10s
      4. 72C - 30s/kb target gene
      5. Repeat 2-4 29x
      6. 72C - 5min
      7. 10C - forever
3. Run a 1% agarose gel of the synthesis and amplification reaction
   1. 5uL sample, 1uL loading buffer
   2. You should see a smear from 60bp to over your gene length in the synthesis reaction
   3. In the Amplification reaction a single band with your gene of interest should be there
4. Continue on with standard cloning!
   1. Make sure to sequence at least 4 clones. Often all 4 will be correct, but insertions,deletions, and spurious mutations sometime occur during the synthesis protocol.
5. TROUBLESHOOTING
   1. Often I focus on the amplification step, assuming that there is a smear for the synthesis step on the gel and that smear covers the size of your gene of interest.
   2. First I often remove the annealing step use a 2 step protocol (Denature – Amplify x 29)!
   3. Still, if no gene is amplified I run a gradient PCR
   4. Then I try 0.5M Betaine (from 5M stock), or 5% DMSO
   5. Finally if nothing is working I break the gene into chunks and amplify smaller sections, then add those sections together and try to amplify the entire gene from the larger chunks.