Sanger sequencing
Keck DNA Sequencing
Monday - Friday, 9 a.m. to 5 p.m.
Sanger sequencing template preparation instructions
- All samples must be submitted premixed, with (1) primer and (1) template in a single tube/well according to the guidelines below. The protocols are sufficient for multiple sequencing reactions, which allows us to repeat a sample in case of technical failure without having to contact you for more sample.
Submission instructions
You may submit in either individual tubes or in plate format. Please see the following pages for more info:
- Tube sequencing
For sample pickup locations and shipping information, please navigate here.
Sanger sequencing template prep instructions
2uL 4uM Primer + DNA(see table) + (sterile H2O, if necessary) = 18uL
Product type Product size DNA (ng) Ordering notes PCR 100-300 bp 40-50 *Can be ordered with Difficult Template or shRNA protocol 300-500 bp 40-160 500-1000 bp 500 Plasmid 1000-1500 Single-stranded M13 or phagemic DNA 200-300 Order as single-stranded BAC and phage lambda DNA 4000 Order as BAC - Each reaction should include 2uL of a 4uM Primer. One primer per reaction.
- The total volume should be 18uL. If extra volume needed, use sterile H2O.
- The protocols above are sufficient for multiple sequencing reactions. This allows us to repeat a sample in case of technical failure without having to contact you for more sample.
PCR cleanup: Please be sure samples are cleaned up prior to submission, not just diluted or desalted. Size exclusion columns are recommended. EtOH precipitation is not recommended, as this does not remove unbound dNTPs and primer.
Available to Yale researchers & external researchers
Core websiteData Return
Standard turnaround time for Sanger services is 1 business day. You can check the status of your order on the sample submission page by selecting CHECK EXISTING ORDERS. You will receive your data through Box.com.
Sanger sequencing
Standard turnaround time for Sanger Sequencing is 1 business day. Yale users will receive priority over non-Yale users. If your samples are RUSH samples, please make us aware of this before we receive your submission.
Sample submission Sample sheet for plate submissionsSpecifications
Sequence analysis is carried out on our Applied Biosystems 3730 XL capillary instruments. The sequencing reactions use fluorescently labeled dideoxynucleotides (Big Dye Terminators) and Taq FS DNA polymerase in a thermal cycling protocol. Using good template and primer, Taq/dye-terminator cycle sequencing will provide 1000 bases of sequence with a 98-99% accuracy (exceptional template-primer combinations will yield up to 1200 bases with 98% accuracy).
Rates
As of 7/1/2024
Service | Internal user | External for-profit user | External non-profit user |
---|---|---|---|
Difficult template surcharge, per sample/well | $2.35 | $3.15 | $3.15 |
Single tube sequencing | $3.50 | $5.00 | $4.85 |
Plate sequencing - 96 well - per well/48 min | $1.99 | $2.75 | $2.75 |
384-well sequencing | $1.85 | $2.50 | $2.50 |
Re-plate samples for sequencing | $10.00 | $15.00 | $15.00 |
Alternative sequencing protocols
In addition to our standard protocol, we have alternative sequencing categories you may select. These alternative protocols cost more than our standard protocol.