Sequencing Service Order
Order manual How to check data
Our company not only utilizes advanced automatic gene analyzer ABI3730XL capable of sequencing various templates such as plasmid DNA and PCR products, but also consists of experts and rich infrastructures to provide reliable data for researchers. Furthermore, using our own gene synthesis techniques, both sequencing and oligo synthesis service can be simultaneously run to get the results in a short time. This is especially the case for primer walking and virus/vector sequence validation.
Features and Benefits
High quality data with accurate results through automated process (Phred Score ≥20, Guaranteed read lengths ≥700bp)
• Free universal primer
Supply of commonly used universal primer capable of being selected upon order
• Working Day: Completion within 48 hours of receiving time
• After completing the service, we will provide 4 types of files (ab1, text, PDF, full text) and reports.
• Samples and primers will be discarded after 1 month. A seperate request is required for a long-term storage
◈ Quality Assurance
Bioneer is the holder of Quality Management System Certificates for the following standards.
▶ Sequencing service list
• Standard Sequencing
Standard Sequencing service analyzes the desired Plasmid/PCR product region using a primer. It is optimized for most of the normal samples.
• Full Length Sequencing (Primer walking)
Full Length Sequencing service analyzes plasmids, long inserts, or PCR products by primer walking. It takes about 1 week in 3 kb. An additional period will be required depending on the size.
After the service is completed, the data is provided in the form of contig file after each single data and assembly. The primer and its design information are sent as a dried form.
• Difficult Sequencing
Difficult Sequencing service aims to analyze templates difficult to be analyzed with standard sequencing such as those having GC/AT rich regions, repeated sequences, secondary structures, etc.
This service is also optimized for Bioneer's long time experiences and technique that utilizes analysis conditions and reagents different from standard sequencing.
▶ Additional service list
Depending on the requested samples, additional services can be chosen.
* All material is serviced by our products.
1) Genomic DNA → PCR → Non-purified PCR product → PCR purification/Agarose gel extraction → Purified PCR product
2) Genomic DNA → Primer design → PCR optimization → PCR → Non-purified PCR product → PCR purification/ Agarose gel extraction → Purified PCR product: Custom Sequencing
A purification process must be done to remove impurities prior to the direct sequencing step of PCR products. This is especially crucial when high-quality data is required to eliminate non-specific bands.
- PCR purification:
This method uses a spin-column method, which is suitable when it is certain that no non-specific band will occur. This service uses our PCR Purification Kit PCR Purification Kit (AccuPrep® PCR Purification Kit).
- Agarose gel extraction:
This method extracts DNA fragments from an agarose gel to acquire only the desired bands, which is suitable when non-specific bands occur in the PCR product. This service uses our PCR Purificaiton Kit (AccuPrep® Gel Purification Kit).
• PCR & Purification
If you send the genomic DNA and specific primers, along with background information such as optimized PCR conditions, product size, etc., we can confirm the size to undergo the suitable purification process after PCR. Primer synthesis service is also available if needed. This service uses our high-fidelity PCR premix (AccuPower® ProFi Taq PCR PreMix)and PCR Purification Kit.
▶ Sample preparation condition
• Sample Preparation
Type Concentration Volume/rxn Purity Plasmid DNA ≥100 ng/µl ≥10 µl A260/A280: ~1.8
PCR product ≥50 ng/µl ≥10 µl Non-purified PCR product ≥ 50 ng/µl ≥20 µ Genomic DNA ≥ 30 ng/µl ≥30 µl
• Primer Preparation
Type Concentration Volume/rxn detail Universal primer Free primers are available to use Specific primer 5 pmol/µl (5 µm) 5 µl Sequencing primer titration conditions: 18~22 mer, TM 50~56°C Ordered primer Sequencing and oligos can be ordered at the same time.
• Sample Enclosure
- Single tube type
When preparing with a 1.5 ml microcentrifuge tube, please specify the sample name as it appears on your order sheet.
- Plate type
When preparing with plates, it is highly recommended to use 96-well plate with V-type bottom with samples placed in the order as shown in the diagram below and seal tightly using a cap to prevent cross-contamination.
▶ Service Price
Cat. No. Service Description Unit Price ($) Note Sequencing Service S-3010-1 Standard sequencing <96 rxns 6.00/rxn S-3010-2 Standard sequencing ≥96 rxns 5.00/rxn S-3010-6 Full length sequencing bp 60.00/kb Additional service S-3010-3 PCR purification ea 2.00/ea S-3010-4 Gel-extraction ea 3.00/ea S-3010-5 PCR & Purification ea 5.00/ea S-3010-7 Custom Sequencing Primer Synthesis mer 0.29/mer Based on 25 nmol
▶ Free Primer
|Primer name||Sequence (5' --> 3')||Length [bases]|
|T7promoter||TAA TAC GAC TCA CTA TAG GG||20|
|T7terminator||GCT AGT TAT TGC TCA GCG G||19|
|T3||AAT TAA CCC TCA CTA AAG GG||20|
|SP6||ATT TAG GTG ACA CTA TAG||18|
|EBV-R||GTG GTT TGT CCA AAC TCA TC||20|
|BGH-rev||CTA GAA GGC ACA GTC GAG GC||20|
|M13F(-40)||GTT TTC CCA GTC ACG AC||17|
|M13R(-40)||CAG GAA ACA GCT ATG AC||17|
|M13F(-20)||GTA AAA CGA CGG CCA GT||17|
|M13R(-20)||GCG GAT AAC AAT TTC ACA CAG G||22|
|pGEX5||GGC AAG CCA CGT TTG GTG||18|
|pGEX3||GAG CTG CAT GTG TCA GAG G||19|
|pQE-forward||CCC GAA AAG TGC CAC CTG||18|
|pQE-reverse||GTT CTG AGG TCA TTA CTG G||19|
|EGFP-C||CAT GGT CCT GCT GGA GTT CGT G||22|
|EGFP-N||CGT CGC CGT CCA GCT CGA CCA G||22|
|RVprimer3||CTA GCA AAA TAG GCT GTC CC||20|
|RVprimer4||GAC GAT AGT CAT GCC CCG CG||20|
|GLprimer1||TGT ATC TTA TGG TAC TGT AAC TG||23|
|GLprimer2||CTT TAT GTT TTT GGC GTC TTC CA||23|
|CMV-F||CGC AAA TGG GCG GTA GGC GTG||21|
|CMV30||AAT GTC GTA ATA ACC CCG CCC CGT TGA CGC||30|
|CMV24||TAT TAG GAC AAG GCT GGT GGG CAC||24|
|Gal4AD||TAC CAC TAC AAT GGA TG||17|
|Gal4BD-F||TCA TCG GAA GAG AGT AG||17|
|Gal4BD-R||TTT CTT TGG AGC ACT TGA GC||20|
|MATCHMAKER3||GTG AAC TTG CGG GGT TTT TCA GTA TCT ACG AT||32|
|pBAD-For||ATG CCA TAG CAT TTT TAT CC||20|
|pBAD-Rev||GAT TTA ATC TGT ATC AGG||18|
|SV40-pArev||CCT CTA CAA ATG TGG TAT GG||20|
|SV40-pAF||AAA TAA AGC AAT AGC ATC AC||20|
|malEF||GGT CGT CAG ACT GTC GAT GAA GCC||24|
|pCold-F||ACG CCA TAT CGC CGA AAG G||19|
|pCold-R||TCC CCG CCA AAT GGC AGG GA||20|
|3AOX||GCA AAT GGC ATT CTG ACA TCC||21|
|5AOX||GAC TGG TTC CAA TTG ACA AGC||21|
|pBabe-F||TGA CCT GGG AAG CCT TGG CT||20|
|pBabe-R||TTG CTG ACT AAT TGA GAT GCA TGC TTT||27|
|V5_Reverse||ACC GAG GAG AGG GTT AGG GA||20|
|pJET1.2F||CGA CTC ACT ATA GGG AGA GCG GC||23|
|pJET1.2R||AAG AAC ATC GAT TTT CCA TGG CAG||24|
|27F||AGA GTT TGA TCM TGG CTC AG||20|
|1492R||TAC GGY TAC CTT GTT ACG ACT T||22|
|518F||CCA GCA GCC GCG GTA ATA C||19|
|800R||TAC CAG GGT ATC TAA TCC||18|
|ITS1||TCC GTA GGT GAA CCT GCG G||19|
|ITS2||GCT GCG TTC TTC ATC GAT GC||20|
|ITS3||GCA TCG ATG AAG AAC GCA GC||20|
|ITS4||TCC TCC GCT TAT TGA TAT GC||20|
|ITS5||GGA AGT AAA AGT CGT AAC AAG G||22|
|LCO1490||GGT CAA CAA ATC ATA AAG ATA TTG G||25|
|HCO2198||TAA ACT TCA GGG TGA CCA AAA AAT CA||26|
|pET-Upstream||ATG CGT CCG GCG TAG A||16|
|pBA-F||ATT GTC TCA TGA GCG GAT AC||20|
|pBH-R||GCG TTA TCC CCT GAT TCT GT||20|
|pBIC_F||CTC ATG AGC GGA TAC ATA TTT G||22|
|pBIC_R||GCC GCA GCC GAA CGA CCG AG||20|
(Address: 8-11, Munpyeongseo-ro, Daedeok-gu, Daejeon 34302, Republic of Korea Bioneer Sequencing Team)
Data such as sequencing reports can be recorded and delivered for free. After receiving the first report, the re-analysis results can be obtained within two days.
- Ab1 file: AS electropherogram file viewable with viewer program
Group A) Data with low QV values which may be improved through re-analysis which can be done free for once.
Group B) No electropherogram; this case may be made from the analyst's mistake, so we will undergo re-analysis free for once. Group C) Sequencing limited by the DNA sequences; re-analysis service will not be available as improvements cannot be made
Group D) Other causes such as primer mis-matches; re-analysis service will not be available as improvements cannot be made
In case of mass ordering, price adjustment is possible. Please consult with your salesman.
The primers can be kept, but Bioneer is not responsible for degradation caused by long-term storage. It is recommended to freeze in several tubes and change it every 1 month for long-term usage.
▶ Sequencing Process Information
1) Material & Instruments
① AllInOneCycler™ 96 well PCR system (Block PCR Instrument, Bioneer)
② BigDye® Terminator V3.1 sequencing kit (Applied Biosystems, USA)
③ ABI3730XL (Applied Biosystems, USA)
① Sequencing PCR Cyclic
<Refer to the table below for the amount of template used in PCR>
• DNA size/ DNA amount
|100~200 bp||5~10 ng|
|200~500 bp||10~20 ng|
|500 bp~1 kb||20~50 ng|
|1~2 kb||50~100 ng|
|≥2 kb||100~200 ng|
|Double strand||200~500 ng|
|Cosmid, BAC||300~600 ng|
|Genomic DNA||2~3 µg|
③ ABI3730XL Running