tool will check if there are repeated sequences (such as a scar sequence) located at the 30-end of the primer and if so, will extend the primer to two bases past the repeated sequence to ensure speciﬁcity. Other future additions will include Tm calculations, GC content, and other relevant primer design speciﬁcations. The vector-speciﬁc primers (AF and BR) are already standardized and do not need to be designed with this tool. Maximizing success rates There are a few additional guidelines to follow to maximize success with this method. First, do not use repeated part sequences (e.g. scar sequences, transcription- al terminators, etc.) on the 30 end of your primer to avoid multiple PCR products. The primer design tool described above will eventually check for this. Second, it is better to PCR-amplify the vector (plasmid backbone) with the smaller part to assemble especially if the part is less than 100 bp since the molar ratio between both fragments should be as close as possible. Third, dilute the template DNA as much as possible to avoid ‘background’ plasmids from being transformed later (between 100 and 500 pmol DNA works well for miniprepped plasmids, while plasmids from the 2009 Parts Registry plate diﬀer in concentration). Primers designed and used in this study All primers were ordered from Integrated DNA Technologies (IDT) unmodiﬁed with standard desalting at the 25 nmol scale. Four primers speciﬁc to the pSB1A2 vector (http://partsregistry.org/Part:pSB1A2) were designed and used in this study. In Figure 2a, the Part A forward primer (AF) is: 50-TTCTGGAATTCGCG GCCGCTTCTAG-30 (speciﬁc to the pSB1A2 non-coding sequence preﬁx+5 bases upstream of the preﬁx). The Part B reverse primer (BR) is: 50-CTAGAAGCGGCCGCGA ATTCCAGAA-30 (reverse complement of the AF primer). In Figure 2b, the Part A forward (AF) primer is: 50-TACT AGTAGCGGCCGCTGCAGGCTTC-30 (speciﬁc to the pSB1A2 suﬃx + 5 bases downstream of the suﬃx). The Part B reverse primer (BR) is: 50-GAAGCCTGCAGCGG CCGCTACTAGTA-30 (reverse complement of Part A forward primer). AR and BF primers for both ﬁgures were customized depending on the parts to assemble or re-engineer. We used 48 bp of homology between parts for assembly reactions as described above except in the example shown in Figure 6 where we used 15 bp. PCR and template DNA for PCR Phusion High Fidelity PCR Mastermix was used for PCR in a 25 or 50 ml reaction volume. Reaction steps were used following the Phusion protocol. An amount of 100 pmol forward and reverse primers were added to each reaction. A volume of 1 ml of a 1:1000 diluted miniprepped plasmid or plasmid extracted from the Registry plate ( 100– 500 pmol total) was added to the PCR reaction for template DNA. PCR was performed in an Eppendorf Mastercycler EP S Gradient thermocycler according to the manufacturer’s instructions with the annealing step of 55 C for 30 s. Eppendorf PCR tubes (0.2 ml) were Nucleic Acids Research, 2010, Vol. 38, No. 8 2627 used for all reactions. Digestion with DpnI after the PCR reaction may reduce background plasmids from being transformed later, but in our experience diluting the PCR template reduces background suﬃciently that DpnI digestion is unnecessary. In most cases, this elimin- ates an extra step in the assembly process. PCR product puriﬁcation and quantitation PCR products were puriﬁed with the Qiagen PCR Puriﬁcation Kit and eluted with 30 ml of molecular grade water. PCR products were quantitated with a Nanodrop (Thermo Scientiﬁc). In-Fusion reaction and transformation In-Fusion reactions were performed using the recom- mended protocol with some extra details and exceptions noted here. A 2:1 insert:vector molar ratio was normally used with 100 ng of vector for two-way reactions and a 2:2:1 insert:insert:vector molar ratio was normally used with 100 ng of vector for three-way assemblies (exceptions noted below). The 10 ml volume consisting of insert, vector, and molecular grade water was transferred into the In-Fusion dry-down reaction tube and mixed by pipetting up and down several times. This reaction was transferred into a 0.2 ml PCR tube and incubated in a thermocycler for 15 min at 37 C followed by 15 min at 50 C. A volume of 30 ml TE Buﬀer (pH 8.0) was added to the tube and mixed by pipetting up and down several times. A volume of 2.5 ml of this mixture was added to one 50-ml tube of Fusion-Blue chemically competent cells thawed on ice. Cells were incubated on ice for 30 min, heat shocked at 42 C for 45 s, and put back on ice for 1 min. A volume of 200 ml SOC media was added to the cells and mixed by pipetting up and down a few times. The one hour incubation time was not necessary when trans- forming plasmids conferring ampicillin resistance, but was required with kanamycin resistance conferring plasmids. A volume of 200 ml of the transformant culture was spread on an LB plate supplemented with 100 mg/ml of ampicillin (without the centrifugation steps in the protocol). This procedure normally gives 30–1000 colonies after overnight incubation at 37 C. Quantitating success and mutation rates with diﬀerent homology and primer lengths The assay described in Figure 3 and results in Tables 1 and 2 quantitated the eﬀect of homology and primer lengths on success and mutation rates. Success rate for each assembly was measured by simply counting the number of ﬂuorescing versus non-ﬂuorescing transformant colonies on LB plates supplemented with 100 mg/ml of ampicillin and 0.1 mM IPTG. Mutation rate for each assembly was determined by sequencing eight plasmids extracted from ﬂuorescing cultures grown from individual clones. Colony PCR Screening for the desired construct was generally per- formed by colony PCR, with the exception of functional