Sanger Sequencing
Sanger sequencing services are provided using the Applied Biosystems 3730xl DNA Analyzer. These machines utilize a one-capillary cycle sequencing process with refined, four-dye fluorescent labeling methods and a real-time scanning detector. The biochemistry of the one-lane sequencing is similar to Sanger-based methods.
The JHU ordering server automatically processes data after electrophoresis is complete and provides quality assessment for each sample. Data is available for download, viewing, and editing.
The service provides standard sequencing primers for several vectors. Custom primers should be provided by the user and premixed with the template. Primers can be ordered through our facility using one of our portals as well
Samples should be provided pre-mixed with primer in 1.5ml tubes (please do not send PCR tubes). In each tube, mix 6 ul of primer and 6 ul of template. Use only one primer per tube. To achieve the best sequencing results, send start with the following concentrations prior to mixing:
- Primer: 1 uM for plasmids, 4 uM for BAC/PAC
- PCR Products: 3.0ng/ul/100bp
- Plasmid DNA: 0.2 ug/ul, with a 260/280 ratio between 1.75 and 1.85. 260/280 ratios above 1.9 or below 1.7 will not work.
- BAC/PAC DNA: 0.5 ug/ul
If using the primers available at DAF, please bring 6 ul of template in a tube and add 6 ul of the appropriate primer when you arrive at our facility. If dropping off at a remote location, please clearly indicate which primers need to be added to the tubes. There is no charge for using our primers.
Samples dropped of in the Blalock building by 4 PM will be ready by 10 AM the following business day. Samples dropped off after 4 PM will be held for processing the next day. Sam day sequencing service is available at a higher fee per sample, with a minimum of 5 samples. For same day sequencing, samples must be ordered on the JHU ordering server and physically present in our facility by 9 AM and the data will released by 5 PM.
Samples should be dropped off in our remote locations by 11 AM. Data release will depend on the pick up schedule of the day concerned. On normal days, samples are picked up in the Asthma and Allergy Building by 11:30 and in CRB I B02A by 3:00 PM, but pickups may vary depending on staffing.
Pricing:
Single pre-mixed template & primer: $6
95 samples in a 96 well plate: $5.50 per sample.
BAC protocol reactions (used on BACs, other large templates or very GC rich templates): $23
Same day service: $19 per sample, minimum of 5 samples
Standard primers are available for you at Blalock 1005.