Gene Synthesis Service Mutagenesis Service Codon Library Mutagenesis Service Gene Cloning Service Vector Information AccuGeneBlock Service Gene

Transcription

1 Gene Synthesis Service Mutagenesis Service Codon Library Mutagenesis Service Gene Cloning Service Vector Information AccuGeneBlock Service Gene Synthesis FAQs User Protocol

2 01 Gene Synthesis Gene Synthesis Service 3 Mutagenesis Service 5 Codon Library Mutagenesis Service 7 Gene Cloning Service 9 Vector Information 11 AccuGeneBlock Service 12 Gene Synthesis FAQs 15 User Protocol 18

3 Gene Synthesis Service Overview Gene is the most cost-effective way to enhance your gene research. You can have your gene cloned in your hand with 100% sequencing data within for most cases. You can save your time and money through Bioneer s Gene Synthesis Service than through conventional way to clone a gene. If you like, our free codon optimization will increase protein expression rates and can enhance protein function. In addition, we can construct virtually any targets of DNA easily. Bioneer also offers Mutagenesis as well as Cloning Services. Bioneer strives to provide the high-qualified synthetic genes at a reasonable price. Our goal is to provide you the best value for your research dollar. Description Empower your research with Bioneer s Custom Gene Synthesis Services. With nearly 20 years of in-house production of raw materials (phosphoramidite, reagents and solvents), oligo, molecular biology research reagent production experience, Bioneer now offers a complete custom gene service including sequence design, codon optimization,, cloning and sequencing. Gene is the most cost effective way to enhance your research. In as little as 2 weeks, you can have your Gene cloned in hand and 100% sequence guaranteed. And with Bioneer s great pricing it can cost less to order a synthetic gene from us than to buy all the kits and reagents necessary to PCR, ligate, clone, grow, purify and sequence your gene of interest. If you like, our free codon optimization will increase protein expression rates and can enhance protein function. In addition our optimization can provide gene targets virtually from any target region of interest. Send us your sequence, Gene ID or an accession number. Let us know what you need and we will deliver! Bioneer also offers mutagenesis as well as cloning and subcloning services. Bioneer strives to provide the highest quality synthetic genes at a great price. Our goal is to always provide you with the best value for your research dollar. With high throughput DNA facilities around the world, Bioneer s daily capacity is unsurpassed. Bioneer is unrivalled in its ability to address the needs of our customers: Whether you need one gene, or one hundred. We respond to your needs personally. Features and Benefits Codon optimization free of charge!: Complimentary codon optimization to enhance protein expression and function 100% sequence guarantee: Individual synthetic genes are confirmed by automatic DNA sequencer (ABI 3730) Value pricing: The best value for your research dollar Application Antibody Construction Antibodies targeted toward specific diseases or targets can be codon optimized for maximum expression in the host organism. Also, an antibody library can be constructed to screen for the most efficient antibody variant. Organism Production Optimization Optimize expression of genes related to resource production to maximize industrial biological production efficiency. Gene Construction Get difficult-to-clone DNA sequences easily and enhance the quality of your research by constructing hypothetical genes. Protein Modification Codon optimization can increase protein expression efficiency, and a mutant library derived from this can yield proteins with increased function. Optimizations include secondary structure removal, and repeat reduction as well as organism optimizations. Procedure Order Submission analysis/ Input Synthesis Gene Synthesis Cloning/Sequencing Product shipment 3

4 Gene Synthesis Service Ordering Information Price Minimum Synthesis Price $ Synthesis Period Delivery Form Cloning Vector Subcloning Price Additional Service $0.39/bp (Normal sequence/<3 kb) Gene Synthesis Service 1-1,500 bp Average 7-12 working days 1,501-3,000 bp Average working days 3,001 bp - Inquire 2 ~ 5 μg of lyophilized plasmid * Low copy plasmid is 1-2 μg pbha Other $ (Gene Cloning Service) Produce high yield of plasmid DNA ($100.00/100 μg) *Low copy vector is exemption Basic vector Add $50.00 & 5 working days Additional charges will apply for gene segments containing complexities such as high or low GC content, repeat sequences or homopolymeric runs. If any of your sequence is found to contain the above listed complexities, you will be contacted by our Gene Synthesis Specialist. When choosing the subcloning service with a commercial vector, a vector purchase cost will be billed separately. Synthesized genes will be sequenced. Only 100% sequence identity product will be shipped. If it s founded as gene which is toxic or inhibit the growth in E. coli cell system, customer can get PCR product or plasmid DNA which is form of mutant (Apply this regulation equally even if checking during the ). If customer would like to order gene for protein expression & purification, customer can get the protein directly through ExiProgen by Bioneer. Quotations and Ordering 1. You can order the gene by on-line or . If the gene length shorter than 3 kb, you can check the quote and order by on-line. If you need codon optimization for gene or need mutagenesis/cloning after gene, you should send the order form by If you want order by , Download the ordering form(s) from the link on web site ( 3. Fill out the form and the form to: geneorder@bioneer.co.kr 4. We will review the order and send you service (final quote and service period) via . Cancellation Fee 50% charge of service bill for cancellation in 5 days. 80% charge of service bill for cancellation after 5 days. Free of charge for cancellation after guarantee duration (Guarantee duration: gene period except delivery) Technical Support Tel , 8515 (Synthetic Biology team) gene-support@bioneer.com GMT +9 AM. 9:00 - PM. 6:00 (Monday - Friday) 5. If you decide to go ahead with the service, please confirm the service by an (geneorder@bioneer.co.kr). 6. Service will be initiated when we send out the service start alert to you. BIONEER 4

5 Mutagenesis Service Description The fastest and most cost effective way to construct a mutant gene for protein structure and functional research or to increase enzyme function is to use our Mutagenesis Service combined with our Gene Synthesis Service. Site-directed mutation such as Point Mutation as well as deletion, insertion mutation services are available. For unsurpassed quality and value pricing, partner with Bioneer today. Features and Benefits 100% sequence guarantee: Individual synthetic genes are confirmed by automatic DNA sequencer (ABI 3730) Value pricing: Order Submission analysis/ Input The best value for your research dollar Synthesis Applications Protein Modification Codon optimization can increase protein expression efficiency, and a mutant library derived from this can yield proteins with increased function. Optimizations include secondary structure removal, and repeat reduction as well as organism optimizations. Antibody Construction Antibodies targeted toward specific diseases or targets can be constructed for maximum expression in the host organism. Also, an antibody library can be constructed to screen for the most efficient antibody variant. Organism Production Optimization Gene Synthesis Cloning/Sequencing Product shipment Optimize expression of genes related to resource production to maximize industrial biological production efficiency. Procedure Order Submission analysis/ Input Synthesis Mutagenesis Cloning/Sequencing Product shipment Ordering Information Accepted Materials Price Order Submission Additional Costs Service Period Additional Service Mutagenesis Service Plasmid, Cultured E. coli cell Inner mutation $200.00/area (-1 kb, In less than continuous 20 bp) End mutation (5 or 3 end) $100.00/area (-1 kb, In less than continuous 10 bp) Add $ as increasing each of mutagenesis Cloning areas Cloning/Sequencing Product shipment (ex. Inner analysis/ mutation Input 2 area: $300.00) With cultured cells $ kb - $50.00/500 bp 5 kb - Inquire - 1 kb Average 5-10 working days 1 kb - 3 kb Average working days 3 kb - 5 kb Average working days 5 kb - Inquire Produce high yield of plasmid DNA ($100.00/100 μg) Proceed with Gene Synthesis Service ($ discount) Price & period can be increased based on the structure, quality of gene. If customer needs to send template DNA, please send it as plasmid DNA (over than ng/μl, vol. 10 μl). If customer sends the wrong sample, 50% of total amount will be charged. So please make sure to send us as checking completely by your side. 5

6 Mutagenesis Service Quotation and Ordering 1. Download the ordering form(s) from the link on web site ( 2. Fill out the form and the form to: 3. We will review the order and send you service (final quote and service period) via If you decide to go ahead with the service, please confirm the service by an Cancellation Fee 50% charge of service bill for cancellation in 5 days. 80% charge of service bill for cancellation after 5 days. Free of charge for cancellation after guaranteed duration (Guaranteed duration: Mutagenesis period except delivery). Technical Support Tel , 8515 (Synthetic Biology team) gene-support@bioneer.com GMT +9 AM. 9:00 - PM. 6:00 (Monday - Friday) 5. Service will be initiated when we receive the raw material from you BIONEER 6

7 Codon Library Mutagenesis Service Description Based on a long-standing experience of gene and mutagenesis techniques, Bioneer provides Codon Library Mutagenesis Service. With the Codon Library Mutagenesis Service, we provide gene library by codon-based mutagenesis in response to codon bias of a species of your interest. You simply select the species where your genes will be expressed and specify the location of codon(s) in the gene sequence along with amino acid name(s) that each codon will designate. Bioneer s service team conducts codon mutation according to the codon usage in the species of your interest. Features and Benefits 100% sequence guarantee: Individual synthetic genes are confirmed by automatic DNA sequencer (ABI 3730) Value pricing Order : Synthesis Submission analysis/ Input The best value for your research dollar Applications Protein Modification Codon optimization can increase protein expression efficiency, and a mutant library derived from this can yield proteins with increased function. Optimizations include secondary structure removal, and repeat reduction as well as organism optimizations. Antibody Construction Antibodies targeted toward specific diseases or targets can be constructed for maximum expression in the host organism. Also, an antibody library can be constructed to screen for the most efficient antibody variant. Organism Production Optimization Optimize expression of genes related to resource production to maximize industrial biological production efficiency. Gene Synthesis Cloning/Sequencing Product shipment Procedure Order Submission analysis/ Input Synthesis Mutagenesis Cloning/Sequencing Product shipment Ordering Information Accepted Materials Price Order Submission Additional Costs Service Period Additional Service Codon Library Mutagenesis Service Plasmid, Cultured E. coli cell $150.00/mutation site (- 1 kb insert) We will provide you special discount for below case. Over 10 quantities: $120.00/mutation site Over 50 quantities: $100.00/mutation Cloning site analysis/ Input Cloning/Sequencing Product shipment With cultured cells $ kb - $50.00/500 bp 5 kb - Inquire - 1 kb Average 5-10 working days 1 kb - 3 kb Average working days 3 kb - 5 kb Average working days 5 kb - Inquire Produce high yield of plasmid DNA ($100.00/100 μg) Proceed with Gene Synthesis Service ($ discount) Mutation site units: the maximum continuous seven codons (DNA: maximum 21 bp). Additional charges will apply for gene segments containing complexities such as high or low GC content, repeat sequences or homopolymeric runs. If any of your sequence is found to contain the above listed complexities, you will be contacted by our Gene Synthesis Specialist. When sending us the template DNA, the minimum quality requirements are as follows; For plasmid DNA, the concentration must be ng/μl (at least 10 μl volume). If you send the wrong sample, 50% of the total cost will be charged additionally. 7

8 Codon Library Mutagenesis Service Quotation and Ordering 1. Download the ordering form(s) from the link on web site ( 2. Fill out the form and the form to: 3. We will review the order and send you service (final quote and service period) via If you decide to go ahead with the service, please confirm the service by an Cancellation Regulation 50% charge of service bill for cancellation in 5 days. 80% charge of service bill for cancellation after 5 days. Free of charge for cancellation after guarantee duration (Guarantee duration: Mutagenesis period except delivery). Technical Support Tel , 8515 (Synthetic Biology team) gene-support@bioneer.com GMT +9 AM. 9:00 - PM. 6:00 (Monday - Friday) 5. Service will be initiated when we receive the raw material from you. BIONEER 8

9 Gene Cloning Service Description Let us perform Order the time-consuming task of Synthesis gene cloning, Submission analysis/ Input subcloning and sequence verification. Bioneer brings years of experience in molecular biology together with state-of-the-art QC to guarantee the quality of custom gene cloning service. Features and Benefits 100% Guarantee Individual custom clones are 100% confirmed by sequencing Value pricing The best value for your research dollar - Automatic DNA Order Submission analysis/ Input Synthesis Applications Protein Modification Codon optimization can increase protein expression efficiency, and a mutant library derived from this can yield proteins with increased function. Optimizations include secondary structure removal, and repeat reduction as well as organism optimizations. Gene Synthesis Cloning/Sequencing Product shipment Antibody Construction Antibodies targeted toward specific diseases or targets can be constructed for maximum expression in the host organism. Also, an antibody library can be constructed to screen for the most efficient antibody variant. Mutagenesis Organism Production Cloning/Sequencing Optimization Product shipment Optimize expression of genes related to resource production to maximize industrial biological production efficiency. Procedure Order Submission analysis/ Input Cloning Cloning/Sequencing Product shipment Ordering Information Accepted Materials Price Additional Costs Service Period Additional Service Gene Cloning Service Plasmid, PCR product, Cultured E. coli cell $200.00/cloning (- 1 kb insert, - 7 kb vector) * Possible to do cloning into the vector provided from Bioneer With cultured cells $50.00 Insert $50.00 / 500 bp (>1 kb insert) 7-10 kb: $ Vector 10 kb - : Inquire 1-8 kb (vector+insert) Average 5-10 working days 8-11 kb (vector+insert) Average working days 11 kb - (vector+insert) Inquire Produce high yield of plasmid DNA ($100.00/100 μg) *Low copy vector is exemption Proceed with Gene Synthesis Service($ discount) Price & period can be increased based on the structure, quality of gene. In the cloning service, if customer requests to purchase the commercial vector by Bioneer, vector purchasing cost will be charged separately. If customer needs to send template DNA, please send it as plasmid DNA (over than ng/μl, vol. 10 μl), purified PCR product (over than 50 ng/μl, vol.10 μl). If customer sends the wrong sample, 50% of total amount will be charged. So please make sure to send us as checking completely by your side. If customer cancels the order during, 50% of total amount will be charged. If customer requests to hold the cloning service due to personal reason, we can hold 1 month at the maximum. After 1 month, order will be canceled automatically & 50% of total amount will be charged. 9

10 Gene Cloning Service Quotation and Ordering 1. Download the ordering form(s) from the link on web site ( 2. Fill out the form and the form to: Technical Support Tel , 8515 (Synthetic Biology team) GMT +9 AM. 9:00 - PM. 6:00 (Monday - Friday) 3. We will review the order and send you service (final quote and service period) via If you decide to go ahead with the service, please confirm the service by an (geneorder@bioneer.co.kr). 5. Service will be initiated when we receive the raw material from you. BIONEER 10

11 Vector Information When using Bioneer s Gene Synthesis Service & Cloning Service, customers can select a vector according to their purpose, which makes them easy to get appropriate plasmid without paying for commercial vector purchase. Please refer to below vector customers can select: High copy vector - pbha, pbhk, pbhc & pbhz Low copy vector - pbla, pblk, pblc & pblz In vitro transcription vector - pbic-a Expression vector - pbt7-n-his, pbt7-c-his, pbt7-n-gst & pbt7-c-gst With the expression vector, customers can check protein expression & purification more easily by using Bioneer s ExiProgen fully automated protein and purification system or AccuRapid manual type kit. Vector Group Vector Name Feature and Antibiotics Marker pbha puc Origin, Ampicillin High Copy Vector pbhk puc Origin, Kanamycin pbhc puc Origin, Chloramphenicol pbhz puc Origin, Zeocin pbla P15A Origin, Ampicillin Low Copy Vector pblk P15A Origin, Kanamycin pblc P15A Origin, Chloramphenicol PBLZ P15A Origin, Zeocin In-vitro Transcription Vector PBIC-A T7 & SP6 Promoter, Ampicillin pbt7-n-his T7 Promoter, N-terminal His-tag, Ampicillin Expression Vector pbt7-c-his T7 Promoter, C-terminal His-tag, Ampicillin pbt7-n-gst T7 Promoter, N-terminal GST-tag, Ampicillin pbt7-c-gst T7 Promoter, C-terminal GST-tag, Ampicillin * vector map:

12 AccuGeneBlock Service Description AccuGeneBlock Service provides of double strand DNA fragments (less than 1 kb) in as little as 3 days. It is a brand new service offered by Bioneer, a leader in Gene Synthesis. AccuGeneBlock Service provides you with your gene fragments at a great price with fast turnaround time so you can use it easily and quickly in synthetic biology research. Features and Benefits Value Pricing The best value for your research dollar ($95.00 for <500 bp). Accurate and Fast Synthesis Fully automatic makes it possible to synthesize highquality genes in as little as 3 days. Easy Protein Synthesis Gene fragments can be used for protein with PCR templates using ExiProgen. Synthesis of Large Genes You may combine multiple AccuGeneBlocks in order to synthesize large genes through cloning. Codon Optimization - Free of charge! Complimentary codon optimization on request to enhance protein expression and functionality. Applications Synthesis of large genes It is possible to make the larger genes by combining multiple AccuGeneBlocks through cloning, gene assembly methods, etc. using the double strand DNA provided. Protein Modification Codon optimization can increase protein expression levels, and a mutant library derived from this can provide proteins with enhanced function. Optimizations include the removal of secondary structure and/or repeat reduction, as well as optimization for expression in different organisms. Antibody Gene Construction Antibodies which target specific diseases or proteins can be codon-optimized for maximum expression in the host organism. Also, an antibody library can be constructed to screen for the best antibody variant. Organism Production Optimization Optimization of gene expression to maximize the efficiency and yield of industrial biological production facilities. Gene Construction Easy production of hard-to-clone DNA sequences. Procedure Order Submission analysis/ Input Synthesis Fragment Product shipment BIONEER 12

13 AccuGeneBlock Service Ordering Information AccuGeneBlock Service Price Service Period Delivery Form Additional Service <500 bp $ ,000 bp $95.00 ( + $29.00/100 bp) <500 bp Average 3-5 working days 501-1,000 bp Average 4-6 working days 500 ng - 1 µg of lyophilized PCR product Increased Quantity 5 μg/$ Codon optimization Free-of-Charge (via ordering only) AccuGeneBlock Service may not be available if the gene sequence contains high or low GC content, repeatitive sequence, homo-polymeric runs, etc. In these instances please use Bioneer s Gene Synthesis Service. Available for gene of <1 kb in size. Double-Stranded DNA with a single 3 A overhang is provided as a default. Blunt-ended DNA is available upon request. Ordering through is required for a gene with codon optimization. Other genes may be ordered Online. All of products will be delivered after checking the sequence. Quotation and Ordering 1. You can order the AccuGeneBlock by on-line or . You can check the quote and order conveniently by on-line. If you need codon optimization, you should send the order form by If you want order by , Download the ordering form(s) from the link on web site ( 3. Fill out the form and the form to: geneorder@bioneer.co.kr 4. We will review the order and send you service (final quote and service period) via If you decide to go ahead with the service, please confirm the service by an (geneorder@bioneer.co.kr). 6. Service will be initiated when we send out the service start alert to you. Cancellation regulation Cancellation will be not allowed since it started. Technical Support Tel , 8515 (Synthetic Biology team) gene-support@bioneer.com GMT +9 AM. 9:00 - PM. 6:00 (Monday - Friday) 13

14 Gene Synthesis FAQs General 1. What does Bioneer provide in the Gene Synthesis Service? We provide 100% sequence-matched gene to our customers in a short time! We have a large capability of oligomer through HT-oligo TM oligonucleotide synthesizer developed by our own technology. That advantage in oligo, along with strong background in molecular biological, makes it possible to generate your gene-of-interest with superior quality in an economical way. 2. What is the difference between oligo and Gene Synthesis? Oligo means chemical of DNA. Gene Synthesis also includes a step of oligo, but it contains additional steps such as analysis of sequences to be synthesized, design and mixing of oligos, ligase chain reaction, PCR and cloning of synthesized gene into a vector. 3. What Gene Synthesis services Bioneer provide to us? We provide Gene Synthesis, Mutagenesis, Gene Cloning, AccuGeneBlock, Rapid Gene Synthesis and Gene-to-Protein Service. 4. What are the applications of Gene Synthesis? Gene Synthesis could be used as useful upstream method of various applications. They include antibody construction, organism production optimization, gene construction, protein modification, point mutation, and gene cloning. Ordering 1. How can I get a quotation of Gene Synthesis Service from Bioneer? You can easily download Bioneer s Standard Order Form GeneSynthesisServiceOrderUSForm.xlsx from our website ( This file could also be used for request of quotation. Please choose what service(s) you want to order, fill it up everything as much as you can, and it to geneorder@bioneer.com. We will provide you quotation, including exact pricing and working period for your gene, in 1 business day (weekend/holidays excluded) typically. 2. Is it helpful to provide sequence about the gene-of-interest? Yes, we can give you exact pricing with the about your gene sequence after thorough analysis of that. 4. Can I get an estimate of Gene Synthesis without of gene sequence? You can refer the following websites to get general idea on pricing of Gene Synthesis. ( GeneSynthesisService-overview.aspx) Please note that pricing and/or working period would be changed if your DNA sequence has higher/lower GC contents, tandem repeats, inverted repeats, and/or homo-polymeric sequences. Additional price, as well as more working period with no guarantee, would also be applied if synthesized gene has been found to be toxic to E. coli or inhibit growth of those organisms. 5. How long does it take for Gene Synthesis Service? When is the starting timepoint? Please refer the website shown in the #4. Regarding the starting timepoint, it is the time we send Confirmation of order to you for Gene Synthesis / AccuGeneBlock / Rapid Gene Synthesis Service. For Mutagenesis / Cloning Service, it is the time your genetic materials arrive at Bioneer. 7. I am fully satisfied with quotation from Bioneer. What should I do to order it? You simply need to reply to our of quotation, telling us that you decided to order it. Our sales representative will contact you shortly. 8. How can I resuspend my gene product? What would be the best storage condition? You can add 20 µl of distilled water or TE buffer to the delivered DNA, with the final concentration of 250 ng/µl. We recommend the solubilized DNA to be incubated at 4 C for 10 min before use. You can store the DNA at room temperature in its lyophilized form. For long-term storage it would be recommended to put the DNA tube in -20 C freezer. 9. Does Bioneer store about my gene and its product after delivery of shipment to me? We store about your gene and its product for 6 months in our storage after delivery. If you want us to dispose it immediately after production, please contact us. Without your consent in written form these would not be disclosed or transferred to anyone. 3.What should I do if I have special, which is necessary for quotation ing but cannot be written on Bioneer s standard ordering form? You can write up that special either in body text of your or in a separated file. BIONEER 14

15 Gene Synthesis FAQs Gene Synthesis 1. What is the maximum length of gene can be synthesized? There is no limitation in its length virtually, but it may take longer time than expected if length is >3 kb. Please contact Bioneer s Gene Synthesis Team for further discussion about it. 2. Is it possible to generate new vector of gene (form of circular DNA)? Yes, it is. But it should be noted that pricing and/or working period will be different from that of linear DNA, because special procedures could be applied. 3. What is the final form product provided to me in Gene Synthesis Service? Your gene of interest will be given as an insertion into our vector, pbha, in 2-5 µg of lyophilized plasmid DNA form. 4. Why additional price/more working period could be applied in case of higher/lower GC contents, tandem repeats, inverted repeats, and/or homo-polymeric sequence? PCR is the most popular way to be used for Gene Synthesis. But the existence of repeated sequence or other barriers often prevents success of Gene Synthesis through PCR. In that case design of more or alternative experimental procedures should be required, which result in additional pricing. 5. Is it possible to clone my gene-of-interest into the vector I choose? Yes, you can also use Bioneer s Gene Cloning Service. Please fill out everything in the third sheet of our standard order form and it to geneorder@bioneer.com. You will get discount of pricing if you use Gene Synthesis Service and Cloning Service simultaneously. 6. Is it possible to do codon optimization with my gene-ofinterest? Yes. Please choose Yes and write exact species in the codon optimization field of our standard order form. 7. What codon optimization program will be used? We are using the codon optimization program which has been developed by Bioneer and KAIST (Korea Advanced Institute of Science and Technology) together. 8. Can I use that codon optimization program by myself? Yes. Please visit the following website ( codon/), then choose the species you want for codon optimization from its list. Please call , or if you want different species other than the list. 10. How much do I have to pay if I cancel my order? You will be charged 50% of your quotation price, if you cancel your order within 5 days after you receive of order from Bioneer. After 5 days of , 80% of your quotation price will be charged. Mutagenesis & Gene Cloning Service 1. What should I provide for Gene Cloning Service from Bioneer? You need to send the following materials; 1) a vector for cloning (plasmid DNA; ng/µl, >10 µl volume), and 2) DNA templates (50 ng/µl, >10 µl volume) which will be used as an insert, through FedEx to the following address; 8-11 Munpyeongseoro, Daedeok-gu, Daejeon, 34302, South Korea Please let us know if you do not have a vector for cloning. We will purchase that vector from its provider for your project, and pricing of that vector will be added to the final quotation. 2. Is it possible to increase quantity of vector with the cloned gene? Yes. Five days and $100 of additional charge will be added per increase of 100 µg of vector with the cloned gene. AccuGeneBlock Service 1. Are there any limitations to gene by AccuGeneBlock Service? It is possible to synthesize the DNA 100 bp - 1 kb size that does not have any of the following characteristics: High or Low GC contents, sequence repeats, or invert sequence(s). 2. What can I do if my gene contains the adverse factors shown in Question 1? We recommend using our Gene Synthesis Service in these cases. 3. What is Toxic? It is DNA sequence of Select Agents and Toxins, which is described in Harmonized screening protocol at International Gene Synthesis Consortium (IGSC). 4. Is it possible to synthesize Toxic? Yes, but you need to submit a consent form from your institute or government that the synthesized Toxic will be used for research purpose only. 9. Can I change gene sequence after my service started? You may change part of your sequence anytime before we complete your order. If that change occurs, you may be charged $50 (less than 9 bp in either 5 - or 3 - end) or $100 (less than 20 bp inside the gene). Please contact us as soon as you need to change the sequence. 15

16 User Protocol Synthetic gene manual Use in when you want to increase 1. Add 20 µl of DW or TE buffer into delivered DNA (Final ng/µl). 2. Prepare competent cell. (Commercial competent E. coli or selfmade competent E. coli. DH5α strain is recommended). Use it when you want to experiment 1. Add 20 μl of D.W. or TE buffer into delivered DNA (Final ng/ µl). 2. After quantitative measurement of DNA, restriction enzyme cut or PCR depending on the purpose and use immediately. 3. Carefully transfer 1 µl of plasmid DNA into the tube prepared in step Place the tube on ice for 20 minutes. 5. Heat-shock the cells for seconds in a water bath at exactly 42 C. 6. Immediately place the competent cell+dna mixture back to ice for 3 minutes. 7. Put the mixture into 10 ml of LB + Ampicillin (50 µg/ml µg/ ml) culture medium. 8. Incubate for overnight at 37 C with shacking (200 rpm, about 16 hr) and isolate plasmid DNA using Accuprep Plasmid extraction kit. 9. Restriction enzyme cut or PCR depending on the purpose and use after quantitative measurement of DNA. Delivery form is lyophilized plasmid DNA cloned in vector (Can be stored at room temperature). After dissolving DNA in D.W. or TE buffer, it is recommended that place DNA at 4 C for 10 minutes. Store at -20 Cafter adding D.W. or TE buffer. BIONEER 16