Team:Washington/Notebook/gene synthesis

Gene Synthesis

 * 1) Generate Oligo's
 * 2) Go to: http://helixweb.nih.gov/dnaworks/
 * 3) Set parameters
 * 4) Enter you job title and email
 * 5) Choose E.Coli Class II for codon frequency
 * 6) Set Annealing temperature to 60
 * 7) Maximize oligo length for cheapest oligo (60 for most companies)
 * 8) Set Number of solutions = 10
 * 9) Select Non-degenerate sites to avoid
 * 10) Bio-Brick requires EcoRI, XbaI, SpeI, and PstI, others can be chosen if desired
 * 11) Leave rest of options default
 * 12) Enter Sequences
 * 13) Click "Add Sequence Field" twice under Sequence formats
 * 14) Imput your header sequence (select Nucleotide, this contains your cut sites,spacers, etc for subsequent cloning)
 * 15) eg. GGATAGGA CATATG
 * 16) Enter you protein sequence (select protein)
 * 17) Imput your tail sequence (select Nucleotide, this contains your cut sites,spacers, etc for subsequent cloning)
 * 18) e.g. CTCGAG ATTCGATG
 * 19) RUN
 * 20) If nothing is running make sure there are no blank new lines in your sequence section!
 * 21) Choose your favorite oligo set to synthesize your gene
 * 22) Usually look for the best scoring with the closest Tm's and oligo lengths
 * 23) Design two additional oligos to amplify your gene
 * 24) 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.
 * 25) ORDER
 * 26) 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.
 * 27) Synthesize Gene
 * 28) Dilute all oligos to 100uM
 * 29) Mix together
 * 30) add 5uL of each into a new master tube
 * 31) Setup Synthesis PCR Reaction (have tried Taq, Vent, and PfuTurbo. Results are always best with Phusion)
 * 32) 1uL Oligo Mix
 * 33) 1uL 25mM dNTP's
 * 34) 10uL Phusion HF Buffer
 * 35) 0.5uL Forward Oligo
 * 36) 0.5uL Reverse Oligo
 * 37) 0.5uL Phusion polymerase
 * 38) 36.5uL diH2O
 * 39) Synthesis PCR Reaction
 * 40) 98C - 30s
 * 41) 98C - 10s
 * 42) 63C - 10s
 * 43) 72C - 30s/kb target gene
 * 44) Repeat 2-4 29x
 * 45) 72C - 5min
 * 46) 10C - forever
 * 47) Setup Amplification PCR Reaction
 * 48) 1uL FROM UNPURIFIED SYNTHESIS REACTION
 * 49) 1uL 25mM dNTP's
 * 50) 10uL Phusion HF Buffer
 * 51) 0.5uL Forward Primer (Tm 65)
 * 52) 0.5uL Reverse Primer (Tm 65)
 * 53) 0.5uL Phusion polymerase
 * 54) 36.5uL diH2O
 * 55) Amplification PCR Reaction
 * 56) 98C - 30s
 * 57) 98C - 10s
 * 58) 63C - 10s
 * 59) 72C - 30s/kb target gene
 * 60) Repeat 2-4 29x
 * 61) 72C - 5min
 * 62) 10C - forever
 * 63) Run a 1% agarose gel of the synthesis and amplification reaction
 * 64) 5uL sample, 1uL loading buffer
 * 65) You should see a smear from 60bp to over your gene length in the synthesis reaction
 * 66) In the Amplification reaction a single band with your gene of interest should be there
 * 67) Continue on with standard cloning!
 * 68) 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.
 * 69) TROUBLESHOOTING
 * 70) 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.
 * 71) First I often remove the annealing step use a 2 step protocol (Denature – Amplify x 29)!
 * 72) Still, if no gene is amplified I run a gradient PCR
 * 73) Then I try 0.5M Betaine (from 5M stock), or 5% DMSO
 * 74) 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.