Sanger Sequencing
SUMMARY OF SERVICES Sequencing
The BMGC DNA Sequencing and Analysis Facility utilizes a capillary electrophoretic DNA analyzer for quick turnaround on small and high-throughput projects. One 96 capillary ABI 3730xl uses ABI BigDye version 3.1 Terminator chemistry.
NEW SANGER SERVICE OPTIONS (details listed below) 1) Sanger High Throughput (Plates): NEW PLATE FORM! Allows clients to name individual samples on a plate. 2) Sanger Classic (Individual) 3) Sanger QC 4) Clean & Load 5) Sanger Custom 6) Difficult Template Panel PRICES & QUICK GUIDE | Service Option | University Rate | Educational Rate | External Rate | Sample Submission | Template, Primer Input | Repeat reaction if failure? | Includes QC? | Primer Bank Option? | Consult with BMGC staff for troubleshooting? | | 1) Sanger High Throughput | $4.11 | $7.11 | $10.00 | 96-Well ABI Fast Plate | 6 ul template + primer | No | No | No | No | | 2) Sanger Classic | $8.09 | $11.09 | $15.00 | 0.5 ml tube | 12 template + primer | Yes | No | No | Yes | | 3) Sanger QC: High Throughput | $9.65 | - | $14.00 | 96-Well ABI Fast Plate | 15 ul template + primer (separate) | Yes | Yes | Yes | Yes | | 3) Sanger QC: Classic | $13.65 | - | $20.00 | 0.5 ml tube | 15 ul template + primer (separate) | Yes | Yes | Yes | Yes | | 4) Clean & Load | $2.95 | - | - | Either 96-Well ABI Fast Plate or 0.5 ml tube | 10 ul completed sequencing reaction | No | No | No | No | | 5) Sanger Custom | $14.57 | $17.57 | $22.00 | Either 96-Well ABI Fast Plate or 0.5 ml tube | 12 ul template + primer (separate) | Yes | Yes | Yes | Yes | | 6) Difficult Template Panel | $163.73 | - | - | 0.5 ml tube | 40 ul template + primer (separate) | Yes | Yes | Yes | No |
HOW TO ORDER
After completing form, print and submit with your samples. You will receive an email after your entire order has been posted. If you have not received an email within 72 hours of order submission, please contact us at bmgcseq@umn.edu and include your order number. If any samples need to be reprocessed, an email will not be sent until the entire order is complete.
SANGER SERVICE DETAILS 1) Sanger HTP (ABI Fast Plates) NEW PLATE FORM! Please fill out and upload the excel spreadsheet for all plate submissions. The new plate form allows clients to name individual samples on a plate. Make sure to follow all directions on spreadsheet. Questions call # 612-625-7736. - Details: Submit template and primer combined in a 96 well ABI Fast plates.
*Plates: can be purchased through facility by submitting an online order for DNA Sequencing Request (Sanger HTP). Please call the lab at 612-625-7736 when completing the online form. The price will be changed in the online ordering system to reflect the plate price of $2.60/plate. A pack of 10 plates can be purchased through www.appliedbiosystems.com, part number 4346907. *Hard copies are not included printing is $1/page. *Minimum number of samples needed for plate submission is 48. If partial plate is submitted, samples need to be loaded from A1-H1, A2-H2, A3-H3, A4-H4, A5-H5, A6-H6, etc. Empty wells must be guaranteed to be clean and empty. Please note this in the additional comments on your DNA sequencing request form. *Controls: You must leave two wells open for our internal control (random well noted on plate and request form).
- Sample submission: 96 well ABI Fast plate and DNA Sequencing Plate Request Form.
- Template and Primer Input: 6 ul.
- Results: No additional repeats, QC, primer banking, or troubleshooting.
Input: Sanger HTP (ABI Fast Plates) | PCR Template | Plasmid Template and quantity to submit | | PCR product size | Quantity to submit | 100 ng per Kb MAX: 800 ng | | 0-200 bp | 1.0 - 3.0 ng | | 200-500 bp | 3.0 - 10.0 ng | BAC, Cosmid, Fosmid, Genomic DNA and quantity to submit | | 500-1000 bp | 10.0 - 20.0 ng | | 1000-2000 bp | 20.00 - 40.00 ng | 0.5 - 3 ug | | >2000 bp | 40.0 - 100.0 ng | | *3.2 pmole of single sequencing primer |
Recommended template and primer concentrations (concentrations may need to be adjusted for each researcher). 2) Sanger Classic (Individual Tubes) - Details: Submit template and primer combined in a 0.5 ml tube.
- Sample submission: 0.5 ml tube and DNA Sequencing Individual Request Form.
- Template and Primer Input: 12 ul.
- Results: No QC, and primer banking. Repeats and troubleshooting are acceptable.
Input: Sanger Classic (Individual) | PCR Template | Plasmid Template and quantity to submit | | PCR product size | Quantity to submit | 100 ng per Kb MAX: 800 ng | | 0-200 bp | 2.0 - 6.0 ng | | 200-500 bp | 6.0 - 20.0 ng | BAC, Cosmid, Fosmid, Genomic DNA and quantity to submit
| | 500-100 bp | 20.0 - 40.0 ng | | 1000-2000 bp | 40.00 - 80.00 ng | 0.5 - 3 ug | | >2000 bp | 80.0 - 100.0 ng | | *6.4 pmole of single sequencing primer |
Recommended template and primer concentrations (concentrations may need to be adjusted for each researcher). 3) Sanger QC - Details: Template and primers submitted separate; may use the primer bank if the primer has been submitted previously.
*Plate submission: BMGC will perform QC. A QC report will be generated and sent to client. Samples that fail QC will be sequenced and charged, results will not be guaranteed for samples that fail QC. Clients will have the option to pick-up plate after QC to remove any failed samples, charges for resubmission will apply.
*Individual submission: We will perform QC and prep samples for sequencing, if sample fails QC then sample will be removed and the online submission form will be updated with changes. A QC report will also be delivered with the sequencing results. Clients have the option to pick-up samples that failed QC. *Any samples that passed QC but failed sequencing will automatically be repeated.
*Submit: Sanger QC form electronically, online submission plate or online submission individual form with samples and primers.
- Sample submission: either 0.5 ml tube or 96 well ABI Fast plate (Plate can be purchased through the BMGC).
- Template and Primer Input: 15 ul template plus primer if not in bank.
- Results: Repeats, QC, primer banking, and troubleshooting are acceptable.
4) Clean & Load - Details: This service allows clients to set-up their own Sanger sequencing reaction. Clients submits reactions ready to be cleaned–up and processed on the capillary electrophoresis instrument.
- Sample submission: either 0.5 ml tube or 96 well ABI Fast plate (Plate can be purchased through the BMGC).
- Reaction Input: 10 ul completed sequencing reaction.
- Results: as-is results there will be no additional repeats, QC, primer banking or troubleshooting.
5) Sanger Custom - Details: Custom includes problematic templates (BACs, Fosmids, giant fragments, high GC, or any other unique situation). Usually a client will choose a protocol to run after completing the “Difficult Template Panel” or if they already know the ideal protocol to use from a previous submission.
*Template and primer submitted separate.
Available Options:
A: DMSO (BMGC adds DMSO)
B: 1 M betaine, 40 cycles, template denaturation
C: 1 M betaine plus dGTP, 40 cycles, template denaturation
D: 5 M betaine, 40 cycles, template denaturation
E: 40 cycles, template denaturation
F: 25 cycles, template denaturation
G: 1 M betaine, 25 cycles, template denaturation
H: BAC/Fosmids
- Sample submission: either 0.5 ml tube or 96 well ABI Fast plate (Plate can be purchased through the BMGC).
- Template and Primer Input: 12 ul template plus primer if not in bank.
- Results: No QC, and troubleshooting. Repeats and primer banking are acceptable.
6) Difficult Template Panel - Details: BMGC provides a panel of alternate protocols. We will run 1-3 samples across the panel to verify the best conditions to use to sequence the difficult template. We will provide a QC report and sequencing report with the recommended protocol to use for future submissions.
*Template and primer submitted separate. Panel:
A: DMSO
B: 1 M betaine, 40 cycles, template denaturation
C: 1 M betaine plus dGTP, 40 cycles, template denaturation
D: 5 M betaine, 40 cycles, template denaturation
E: 40 cycles, template denaturation
F: 25 cycles, template denaturation
G: 1 M betaine, 25 cycles, template denaturation
H: BMGC standard
- Sample submission: 0.5 ml tube and DNA Sequencing Individual Request Form.
- Template and Primer Input: 40 ul template plus primer if not in bank.
- Results: QC, primer banking, repeats and troubleshooting are acceptable.
General guidelines for all samples
Primers Primers used for sequencing are best between 18-22 bases in length with a predicted Tm between 50-55ºC. G-C content of 40-60% is desirable. Avoid primers with strong self-dimers or hairpins. Also, avoid primers which are >90% homologous to other regions within your sample. A BLAST search will alert you to this problem. The best sequencing data is found 80-150 bases away from the primer. It is difficult to get good basecalling less than 50 bp away from the primer. Only sequence in one direction. Submit forward and reverse primers in separate tubes. Primers may be ordered through the Oligonucleotide and Peptide Synthesis Facility. For information regarding primers, go to Integrated DNA Technologies' website
How to clean-up samples - PCR samples we recommend Qiagen PCR purification kit or Microcon filters.
- Plasmid samples we recommend Milipore Qiagen mini-prep columns or Qiagens QIAprep Spin Miniprep kit.
- BAC and cosmid we recommend Qiagen's products
- Genomic DNA we recommend Promega Wizard Genomic kit
Where can samples be dropped-off? We have 3 drop-off locations - Minneapolis campus drop-off sites: 1-202 Nils Hasselmo Hall - Map (samples are picked up at 9:45 AM and 1:45 PM)
- 3rd Floor Lab Elevator Medical Biosciences Building - Map to Medical Biosciences Building (MBB)
- St. Paul campus: 20 Snyder Hall - Map
Sending samples – mailing address Sending samples – shipping address
Where can results be obtained? Results are in two different formats: text (.seq files) and trace (.ab1 files). - University researchers data is posted on the online ordering system at https://www.ahcs.umn.edu/bmgc/index.cfm; go to "My Account" tab and then the "Result Files" section on the right next to "History".
- Researchers outside the University data will be emailed to the email address provided on the DNA sequencing request form.
What is the turnaround time? We have a 72-hour turnaround time. If you have not received an email message before the 72-hour turnaround time, please contact us at bmgcseq@umn.edu and include your order number.
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