Sanger Sequencing Services:
- Sanger-based DNA sequencing
- Sanger-based gene resequencing
Learn more about each of these services below:
Sanger-based DNA Sequencing
The ATGC provides DNA Sequencing from single stranded or double stranded DNA, from purified plasmids, PCR products, and BACs.
The ATGC provides DNA Sequencing from single stranded or double stranded DNA, from purified plasmids, PCR products, and BACs. Sequencing is performed primarily on ABI 3730XL and 3730 DNA sequencers using Big Dye terminator cycle sequencing chemistry. The facility quantifies all samples, performs sequencing reactions, cleanup and capillary electrophoresis, analyzes the data and provides sequence as text files and chromatograms.
View our service pricing schedule for more information about DNA sequencing pricing.
Turnaround Time: 24-48 hours (weekdays)
Longer turnaround times may occur when our sample volume is very high, when special conditions are requested and when we experience instrument problems.
Guidelines for DNA submission
Sample submission requirements
Plasmid concentration must be 100 ng/µl. Submit 10 µl per reaction. Custom primers must be at 1 pmol/µl. Submit 10 µl per reaction. The DNA and primer must be submitted in 0.5-ml Eppendorf tubes with the sample name written on the sides and tops of the tubes. BAC DNA should be submitted at 500 ng/µl and primers at 25 pmol/µl. PCR products should be submitted at a minimum concentration of 20 ng/µl for products less than 1 kb. Products 1 kb or greater should be submitted at 30 ng/µl. Submit 10 µl per reaction
Quantitation of DNA
All DNA submitted to the facility needs to be accurately quantified. We recommend visual determination of DNA quality and quantity on an agarose gel using a quantitative DNA ladder. Alternatively DNA concentration can be determined fluorometrically. DNA concentrations determined using a spectrophotometer are often artificially high due to the presence of RNA, proteins, bacterial genomic DNA and other contaminants.
Note: Low DNA concentration is the most common cause of poor quality sequence and failed reactions. Too much DNA can, however, be as bad as too little. The presence of too much template results in top-heavy data (strong peaks at the beginning which fade rapidly), pull-up peaks (non-specific peaks that appear below the correct peak) and loss of peak resolution. In addition, it shortens the life of our capillaries.
Custom sequencing primer design guidelines
- Thermal cycling conditions: Denaturing step heats the reaction mix to 96°C. The annealing step cools the reaction mix to 55°C, and the polymerization step extends at 60°C. Primers must have annealing temperatures of 56°C or higher.
- Primer length should be at least 20- to 25-mers. GC content should be 50% or more.
- Primers should be designed with a tightly binding 3' end.
- When designing a primer, do not pick a region that is closer than 50 bases to the region of interest.
- Primers for PCR reactions tend to work fine for automated sequencing.
Sequencing primers provided by ATGC
The facility currently provides the following primers free of charge:
- coreT3: CCT CAC TAA AGG GAA CAA AAG C
- coreT7: TAA TAC GAC TCA CTA TAG GGC GA
- coreT30: ATT AAC CCT CAC TAA AGG GA
- coreT70: TAA TAC GAC TCA CTA TAG GG
- coreM13F: GTA AAA CGA CGG CCA G
- coreM13R: TCA CAC AGG AAA CAG CTA TGA C
- coreSp6: GAT TTA GGT GAC ACT ATA G
- coreBluescriptKS: TCG AGG TCG ACG GTA TC
- coreBluescriptSK: CGC TCT AGA ACT AGT GGA TC
- coreBGHRev: TAG AAG GCA CAG TCG AGG
- corePCMVFor: CGC AAA TGG GCG GTA GGC GTG
- corepGEX3: CCG GGA GCT GCA TGT GTC AGA GG
- corepGEX5: GGG CTG GCA AGC CAC GTT TGG TG
- coreT7Ter: GCT AGT TAT TGC TCA GCG G
- corePolyA: AAA AAA AAA AAA AAA AAA AAA AAA T
- corePolyT: TTT TTT TTT TTT TTT TTT TTT TTT A
- coreU6: GAG GGC CTA TTT CCC ATG ATT
The facility will repeat a sample at no cost to the investigator if there is an instrument failure, or if the quality of sequence is compromised due to an error in the facility. If the investigator recommends a sample be repeated, the same DNA and primer will be used to repeat the sequencing reaction. If the reaction fails a second time, the investigator will be charged for the repeated reaction. If an incorrect primer has been requested for sequencing and as a result no sequence data is produced by the sequencing reaction, the cost will be borne by the investigator.
The facility will assist investigators in designing primers to difficult regions. Please contact Erika Thompson if you require this service
Frequently asked questions
View answers to frequently asked questions such as: Why did my template not sequence? Why should I resuspend in water? Does the host strain matter? Does it matter which media I use to grow my cells? Why did my sequence stop short?
*Note: The text sequence provides you with unedited, raw sequence data. Please view the chromatograms to correct minor errors made by the base-calling software. The Sequencing and Microarray Facility provides a server for the rapid dispersal of data to the principal investigators. The results remain on the server for 30 days, after which files will automatically be deleted. Investigators should copy all data to their drives.
When using AppleTalk, the server will only allow 20 users to log on at a time. The server has been set up to allow 15 minutes per user log on to download files. If your workstation logs on automatically at start up you will be bumped off the system after 15 minutes.
(If you do not have a folder online, please contact the ATGC and we will have a folder created for you.)
New Directions to Access Your Online Sequencing Results
- Click "Start"
- Click "Run"
- Type in “\\mymdafiles\seq”
- Click "OK"
- A dialog box will appear. Type in your "username" and "password".
- Click on "Go" (located at top of screen)
- Click on "Connect to Server"
- Type in “smb://mymdafiles/seq”
Both PC and Mac users will use the same username and password that you use to access your MD Anderson account through Entourage and/or Outlook.
Viewing chromatograms (free downloadable software)
Chromas Software: http://technelysium.com.au/wp/chromas
4peaks Software: http://downloads.nucleobytes.com/4peaks
Sanger-based Gene Resequencing
Sanger-based gene resequencing enables gene mutation detection by evaluating an entire gene or individual SNPs in a single experiment. The ATGC performs this assay using internally designed primers. View the genes available for this service.
View the service pricing schedule for more information about gene resequencing pricing.
- Customization available - choose the entire gene, a specific hotspot or a single exon in most cases.
Quantification is performed on a Qubit fluorometer. Sequencing is performed on a 3730XL DNA Analyzer (Thermo) and comparative analysis uses SeqScape software (Thermo).
- Quantification and normalization of samples
- PCR amplification and purification
- Sanger sequencing in both directions (where possible)
- Comparative alignment to the reference sequence
- Re-analysis (if needed)
- Report of all mutations found and all sequences generated sent to customer
Send completed submission form to D.J. Doss or submit hard copy with samples. Samples may be submitted in 1.5 or 0.5 microcentrifuge tubes with the name clearly written on the top of the tube. The amount of gDNA needed is dependent on the gene requested, please contact D.J. Doss for more information.
Scheduling sample drop-off
Contact D.J. Doss to schedule sample drop-off and to determine the amount of gDNA needed.