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=== U. Hawaii BioBrick Primer Design Procedure === ==== 1. Assess BioBrickability ==== * Assess gene BioBricking feasibility. Analyze sequence for existing BioBrick sites to see if additional processing is needed before designing BioBricking primers. [http://syntheticbiology.org/BioBricks/Part_fabrication.html#These_sites_MUST_be_absent site removal reference] : '''These_sites_MUST_be_absent''' [http://syntheticbiology.org/BioBricks/Part_fabrication.html#These_sites_MUST_be_absent] :: <pre>EcoRI, XbaI, SpeI, PstI, NotI</pre> : '''Strongly prefer that these sites be absent''' [http://syntheticbiology.org/BioBricks/Part_fabrication.html#Strongly_prefer_that_these_sites_be_absent] :: ApoI (probably impossible because it occurs frequently), MfeI, AvrII, NheI, NsiI, SbfI : '''Would prefer that these sites be absent''' [http://syntheticbiology.org/BioBricks/Part_fabrication.html#Would_prefer_that_these_sites_be_absent] :: Offset cutters :: <pre>AarI, AcuI (medium priority), BbsI, BbvCI, BciVI (would be nice, medium priority), BfuAI (high priority), BmrI (high priority), BsmBI, BsaI, BsgI (medium priority), BsmI (includes nicking enzyme, high priority), BspMI, BsrDI (includes nicking enzyme, high priority), BtgZI, EarI, EcoP15I (high priority), FokI (best effort), Nt.BstNBI, SapI (high priority, should already be eliminated from EarI), TspRI (probably difficult, best effort)</pre> :: Nicking enzymes :: <pre>Nt.BstNBI, BbvCI, Nt.AlwI (best effort to at least remove sites near each other)</pre> :: Homing endonucleases :: <pre>I-CeuI, I-SceI, PI-PspI, PI-SceI, I-PpoI</pre> :: Others :: <pre>AgeI, AscI, FseI, RsrII, SgrAI, XmnI, XcmI</pre> : '''Would be convenient if these sites were removed''' [http://syntheticbiology.org/BioBricks/Part_fabrication.html#Would_be_convenient_if_these_sites_were_removed] :: These are common, efficient cutters that people might want to use. :: <pre> HindIII, BamHI, XhoI, NcoI, SacI, NdeI </pre> :: Here are other low priority cut sites to remove :: <pre>KasI, MssI, NgoMIV, PacI, PmeI, SalI, SfiI, SgfI, SmiI, SrfI, SwaI, XmaI, ZraI</pre> : '''Sites to include''' [http://syntheticbiology.org/BioBricks/Part_fabrication.html#Sites_to_include] :: Having GATC sites in your part can sometimes be useful because it allows plasmid purified DNA to be cut (via DpnI) whereas PCR product DNA is not. Such a trick is useful for some site directed mutagenesis protocols. : '''Miscellaneous''' [http://syntheticbiology.org/BioBricks/Part_fabrication.html#Miscellaneous] :: BaeI is an enzyme whose is exact cut position is unknown. It is explicitly included in some pSB plasmid replications origins so that the plasmid can optionally be destroyed via another enzyme. You may want to eliminate this site from your part if you intend to use this feature of the BioBricks plasmids. ==== 2. Make Primers ==== * Pick first ~20 and last ~20(reverse complement) residues of full length desired region for making primers, adjust cut length at 3' boundry according to calculated Tm. (target for 60C Tm for first-cycle annealing to your target DNA). Account for necessary adjustments such as replacing stop codon with TAATAA (where replaced region no longer match/anneal to template) . Only calculate Tm for region that is annealing to target site in the first cycle. ** we would like to standardize Tm calculation algorithm used by all team members. Please use:[http://scitools.idtdna.com/analyzer/Applications/OligoAnalyzer/ IDT OligoAnalyzer] with the parameters: Oligo content of 0.4 uM, Na+ concentration of 50 mM, Mg++ concentration of 2mM, and a dNTP concentration of 2mM. ==== 3. Attach BioBrick Ends ==== * Attach biobrick ends accordingly, using the table below. Calculate the primer+extension Tm. ===== Back-Insert Primers (X-SP, preferred) ===== ====== Prefix [X] ====== <pre> EcoRI NotI XbaI GAATTC GCGGCCGC T TCTAGA G regular cct T TCTAGA G [anything] protein (A=overlaps XbaI A, T=spacer, G=n/a) cct T TCTAG [ATG] fusion protein (skips spacer to be in-frame) cct T TCTAGA [no start ATG] edinbricks? </pre> ====== Suffix [SP] ====== <pre> PstI NotI SpeI CTGCAG CGGCCGC T ACTAGT A regular aagg CTGCAG CGGCCGC T ACTAGT A [anything] protein aagg CTGCAG CGGCCGC T ACTAGT A [TTA TTA double stop codon] fusion protein (skips spacer to be in-frame) aagg CTGCAG CGGCCGC T ACTAGT [no stop codon] edinbricks? </pre> ===== Front-Insert Primers (EX-S, not preferred) ===== ====== Prefix [EX] ====== <pre> EcoRI NotI XbaI at GAATTC GCGGCCGC T TCTAGA G regular at GAATTC GCGGCCGC T TCTAGA G [anything] protein (A=overlaps XbaI A, T=spacer, G=n/a) at GAATTC GCGGCCGC T TCTAG [ATG] fusion protein (skips spacer to be in-frame) at GAATTC GCGGCCGC T TCTAGA [no start ATG] edinbricks? </pre> ====== Suffix [S] ====== <pre> PstI NotI SpeI CTGCAG CGGCCGC T ACTAGT A regular c T ACTAGT A [anything] protein c T ACTAGT A [TTA TTA double stop codon] fusion protein (skips spacer to be in-frame) c T ACTAGT [no stop codon] edinbricks? </pre> ===== Back-Insert De Novo Synthesis [prefix]-[gene]-[suffix] ===== Forward Strand: 5'-3' <pre> de novo synthesis regular CTAGA G [anything ] T ACTAGT A GCGGCCG CTGCA - - -------------- - ------ - ------- - de novo synthesis protein CTAG [ATG-----TAATAA] T ACTAGT A GCGGCCG CTGCA -------------- - ------ - ------- - de novo synthesis fusionbricks CTAGA [nostart-nostop] ACTAGT A GCGGCCG CTGCA - -------------- ------ - ------- - de novo synthesis endinbricks </pre> Reverse Strand: 5'-3' <pre> de novo synthesis regular G CGGCCGC T ACTAGT A [anything ] C T ----- ------- - ------ - -------------- - ----- de novo synthesis protein G CGGCCGC T ACTAGT A [TTATTA-----CAT] ----- ------- - ------ - -------------- ---- de novo synthesis fusionbricks G CGGCCGC T ACTAGT [nostart-nostop] T ----- ------- - ------ -------------- ----- de novo synthesis endinbricks </pre> ==== 4. Error Checking ==== # use [http://scitools.idtdna.com/analyzer/Applications/OligoAnalyzer/ IDT OligoAnalyzer] #* hairpins #* self-dimer #* hetero-dimers === Tools === * IDT OligoAnalyser [http://scitools.idtdna.com/analyzer/Applications/OligoAnalyzer/IDT OligoAnalyzer] ** Oligo content of 0.4 uM ** Na+ concentration of 50 mM ** Mg++ concentration of 2mM ** dNTP concentration of 2mM * Alternative Tools *# [http://bioinfo.ut.ee/oligotm/ oligoTM] (web interface to primer3 package's oligotm algorithm) *# [http://www.promega.com/biomath/calc11.htm Nifty alternative tool] to calculate Tm for oligos (thanks Grace!) === Design Notes === * [[Team:Hawaii/Norman's Primer Design Notes|Norman's Primer Design Notes]] * [[Tean:Hawaii/Adam's Primer Design Notes|Adam's Primer Design Notes]] === References === * [http://hdl.handle.net/1721.1/21168 Idempotent Vector Design for Standard Assembly of BioBricks] by [http://openwetware.org/wiki/Tom_Knight Tom Knight] * [http://openwetware.org/wiki/Cfrench:bbprimerdesign BioBrick Primer Design Notes by Chris French] * [http://partsregistry.org/cgi/htdocs/Assembly/rbs_cds.cgi RBS & Protein Coding Sequence biobricking primer design considerations] * [http://syntheticbiology.org/BioBricks/Part_fabrication.html Syntheticbiology.org Biobrick Part Fabrication notes] * [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp NEB: Number of extra spacer nucleotides needed for efficient restriction digestion]

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