Make sure to check out our Back to Work guide and familiarize yourself with all of the new protocols for dropping off/picking up samples, changes to turn around times and more.
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. Please note that Genesifter is only accessible within the Hopkins environment. To access Genesifter from a remote location outside the JHMI domain firewall, log in to your JHED account and follow the instructions in the sidebar VPN link. Once connected through Pulse Secure you will be able to access Genesifter using the link above.
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 and PCR products, 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 indicate which primer should be added. Please clearly indicate which primers need to be added to the tubes. There is no charge for using our primers.
Tubes should be labeled with the order tracking number and the number indicating the sample’s position in the order, i.e. if it is the first sample in the list, place the tracking id and the number 1 on the lid of the tube. If submitting 24 or more samples, you may put them into a 96 well plate PCR plate (in column order) and label the plate with the tracking ID.
Samples dropped of in the Blalock building by 5 PM will be ready by 1 PM the following business day. Samples dropped off after 5 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:
- Blalock 10th floor fridges (near the Ross bridge or in Blalock 1036): 5 PM.
Bayview Asthma & Allergy building 1st floor hallway, 24/7 system, slot F63: 11 AM
- Homewood: Hand to GRCF staff Fridays between noon & 1 PM. Call (410)955-2836 to arrange pickup.
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
- Single pre-mixed template & primer: $5.50
- 95 samples in a 96 well plate: $5.00 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 will be added by our staff. Please indicate which primers are needed on the order form.Order now