DNA Sequencing
The Highest Standard in DNA Sequencing Services
Features
- Sanger DNA Sequencing -Fluorescent dye-terminator sequencing performed on 3730XL sequencers.
- Highest Quality -Sequence length up to 800 bp per reaction, >90% successful rate, >650 bp Q20 read lengths typical.
- Shortest Turnaround -24 hr to 48 hr.
- Very competitive pricing -Special prices on large projects (>100 samples) and contracts.
- Various Samples Acceptable -Samples are accepted as bacterial colonies, glycerol stocks, pelleted cultures and crude PCR products.
- Plentiful Sample Preparation -Plasmid DNA preps from glycerol stocks in advance of sequencing for FREE.
- Universal primers free of charge -Universal primers provided at no additional charge (complete list).
- No penalties -We understand that not everything works out perfectly, samples may be compromised during transit or last minute checks of samples indicate poor quality. We do not charge for reactions we do not perform.
- Free shipping for more than a 100 reactions.
More Information
- Prices
- Sample Requirements and Checklist
- Sequencing Reports Instruction
- Frequently Asked Questions
- Sequencing Terms & Conditions
How To Order
1. Download Sequencing Order Form and email to sequencing@vip.163.com and copy orders to order@sangon.com
2. Send samples to: Sangon Biotech (Shanghai) Co., Ltd. 698 Xiangmin Road, Songjiang District,Shanghai China 201611
We accept plasmid DNA (100 ng/ul, 20 ul), bacteria harboring the target plasmid, PCR products (crude or purified).
Note: Please specify vector name, insert length and resistance.
PCR (crude): concentration > 30 ng/ul, volume >50 ul
PCR (purified): concentration > 30 ng/ul, volume > 20 ul
Note: PCR sample, please indicate the length of product (bp)
Please indicate the concentration of the primer enclosed with sample.
Samples are permanently destroyed after one months.
Checklist Before Sending Samples
Purified PCR Product
1. Dissolve final PCR product in ddH2O (Please do not use TE).
2. Concentration of PCR product should be >30 ng/ul and volume >10 ul. For PCR product >2kb, more DNA may be required for sequencing.
3. A single distinct band of final PCR product should be detected on gel. If smearing or multiple bands are seen, you may receive poor or no readable sequencing data.
4. Specific primers are to be provided by customer.
5. To avoid cross contaminations during shipment, please make sure you use a tight-fit lid when shipping samples in 96-well format. Alternatively, samples can be delivered in 8-strip PCR tubes.
Purified Plasmid DNA
1. Dissolve purified plasmid DNA in 30~50 ul ddH2O (Please do not use TE Buffer).
2. Concentration of plasmid should be >100-200 ng/ul.
3. It is recommended to use molecular biology kit to purify plasmid DNA rather than ethanol precipitation.
4. Specific primers are to be provided by customer.
5. To avoid cross contaminations during shipment, please make sure you use a tight-fit lid when shipping samples in 96-well format. Alternatively, samples can be delivered in 8-strip PCR tubes.
Bacteria harboring plasmids
1. Please indicate antibiotic resistance for desired bacteria strain. Sangon Biotech offers services of bacteria growth with Amp, Kan, Tet and Chl resistance. All other antibiotics are to be provided by customer with instructions.
2. Sangon BiotechInc offers DNA extraction from high copy plasmid. For low copy plasmid bacteria, please send ~10 ug purified DNA.
3. Please provide 1 mL overnight culture or 15% glycerol stock. Please send bacterial samples in individual Eppendorft tubes with openings securely sealed.
4. For large sample size, we recommend E. coli stab in 96-well plate. This is to avoid cross contamination between samples during shipment.
Specific Primers
1. Concentration of primers should be >10 pmol/ul(or 10 uM) and volume > 1-2ul per sample. Please mark primer concentration clearly.
2. Random primers and/or primers <15 bases cannot be used for sequencing.
3. Please provide sequences of primers so that Tm could be calculated for condition optimization. Universal Primers M13 Forward, M13 Reverse, pGEX 3', pGEX 5', T7promoter, T7 terminator, SP6, T3 are primers provided at Sangon Biotech FREE of charge.
If your primers are not in the above list, please ship primers along with your sample(s). Alternatively, Sangon Biotech can synthesize the oligos for you at $0.16 per base. Please refer to Oligo Synthesis.
1. Sequencing report format
(1) Original ABI format of ~700 bp sequencing chromatogram
(2) Summary of Report
(3) All results are sent electronically
2. View results
(1) Download Chromas from www.sangon.com
(2) Double click Chromas and open ABI file from Chromas
(3) Export text file from Chromas
(4) Sequences between 60 bp to 500 bp are most reliable region. To obtain reliable reads, it is highly recommended to perform double stranded sequencing using reverse primers as a good laboratory practice.
3. No report
(1) Samples fail to pass incoming QC
(2) Sample with no readable signal
4. Charges on sequences with structures
Standard charge applies even if sequences are not "clear" due to known structures including Poly N, High or Low GC content, inverted repeats, direct repeats etc.
5. Charges on mixed peaks
Most "mixed" peaks are results from "mixed" templates (see below Q&A). Standard charge applies.
Sample Storage
All samples are kept at Sangon Biotech for 1 month. During this period, customer may request additional sequencing service. After 1 month, the samples are automatically destroyed without further notification.
Q: In one of the region, the sequencing was not clear. Could I request Sangon Biotech to sequence again?
A: Please indicate clearly which position is unclear to you and then we would go case by case. If it is from Sangon Biotech's processing problem, we will initiate a no-charge repeat immediately.
Q: I lost all data, could you send me another copy?
A: Yes. However, please note, all sequencing results are kept for a period of 6 months. Samples are kept at Sangon Biotech for 1-2 months.
Q: I have a 4kb PCR fragments, can Sangon Biotech sequence through the full length?
A: For PCR >2kb, we recommend to first subclone into a vector. The insert is more stable in a vector and sequencing results will be easier to achieve.
Q: I have a mixed base pair in a position, how come I do not see the mixed base in the report?
A: When preparing the report, Sangon Biotech assumes each sequencing sample is from a pure single population rather than mixed. Based on this assumption, the higher/stronger peak is being reported and lower/weaker peak being treated as background. If your sample contains a mixed population, please kindly notify us.
Q: The bands are present and strong, but irregularly spaced, or with mixed colors. The technician may have reported "superimposed sequences" or used the phrase "peaks on peaks"?
A: If you see this, you usually have two sequences superimposed on each other. There are several common causes:
- The sequencing primer binds to two (or more) sites on the template.
- There are two (or more) templates present.
- This was a PCR reaction, and you didn't remove the original primers.
- This was a PCR reaction, and one primer generated *both* ends.
- This was a PCR reaction, and there is more than one amplified species present.
Q: Your sequence proceeds normally, then the bands abruptly become much smaller
A: Secondary structure in the template is the most likely cause of this problem. The polymerase is presumably unable to progress through some stem-loop form. A couple possible solutions: (i) try resequencing by selecting 'siRNA construct' as your DNA type, (ii) try to sequence from another primer at a different position (closer or further); (iii) sequence the other strand.
We maybe able to use special cycling conditions and/or special reagents that help the polymerase to push through this region. We cannot do this routinely, as it involves extensive optimizations.
Q: Your sequence proceeds normally, then the bands abruptly vanish.
A: This usually happens when the template DNA has simply stopped, for example if it was restricted at a downstream site or if the template was a PCR product. This may also be caused by an extremely stable secondary structure.
Q: The sequence looks great until it hits a polyA (or polyT), and then the bands rise and fall in waves.
A: This is called "polymerase slip". It happens when the growing strand temporarily dissociates from the template, then reassociates at a different spot - say, one nucleotide forward or back from where it started. If this happens often enough (as it will on polyA or polyT templates), every individual band becomes a family of closely-spaced peaks giving a 'roller coaster' look to the chromatogram. Try sequencing in the other direction from the opposite strand, or try another primer either closer or further from the homopolymer region.
Q: You offer 24hr to 48hr service, but it has been 2 days and I still have not received data?
A: 24hr to 48hr is AFTER we receive the samples at Sangon Biotech If you have more than 5 plates, longer turnaround is expected.