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.
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
- Purified 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
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.
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. Same 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.
Sample drop off times are as follows:
- Bayview Asthma & Allergy building 1st floor hallway, 24/7 system, slot F63: 11 AM
- Homewood: 12 Noon
- UTL Rm 384 (Black refrigerator in the break room right across from the main staircase.)
- Croft Hall Suite 100 copy room (Black refrigerator next to the copy machine. Please note this fridge is for DNA only.)
- Call 410-955-2836 for the combination to unlock the refrigerators.
- CRB1 Rm B02A 24/7 system, slot A63: 3 PM
Data release will depend on the pick up schedule of the day concerned. On normal days, samples are picked up in the Asthma & Allergy Building by 11:30 AM, Homewood locations by 1:30 PM and in CRB I B02A (slot A63) by 3:30 PM, but pickups may vary depending on staffing.
- Single pre-mixed template & primer: $6.25
- 95 samples in a 96 well plate: $6.00 per sample.
- BAC protocol reactions (used on BACs, other large templates or very GC rich templates): $22
- Same day service: $25 per sample, minimum of 5 samples
Standard primers are available for you at Blalock 1005.Order now