Services Core/facility: Keck DNA Sequencing Facility

Sanger sequencing

Keck DNA Sequencing

Office hours:

Monday - Friday, 9 a.m. to 5 p.m.

Building:

300 George Street, floor 2, room 2127

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:

Sanger sequencing template prep instructions

 
  • 2uL 4uM Primer + DNA(see table) + (sterile H2O, if necessary) = 18uL
  • Product typeProduct sizeDNA (ng)Ordering notes
    PCR100-300 bp40-50*Can be ordered with Difficult Template or shRNA protocol
    300-500 bp40-160
    500-1000 bp500
    Plasmid  1000-1500
    Single-stranded M13 or phagemic DNA  200-300Order as single-stranded
    BAC and phage lambda DNA 4000Order 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 website

Data 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.

Sample Submission 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 submissions

Specifications

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

 
ServiceInternal userExternal for-profit userExternal 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. 

 

 

The Difficult Template Protocol might be useful in achieving better sequencing results if your sample falls into any of these categories:

  • Is GC-rich: above 60% GC or concentrated GC content in a small section
  • Contains secondary structures
  • Has a higher melting temperature/harder to denature
  • Has certain di- or tri-nucleotide repeats

    Prepare sample according to your product type & size. Order as "Difficult Template".

Only high-purity templates should be used to optimize the amount of DNA loaded onto the sequencer.

Recommended BAC template preparation methods:

  1. Alkaline lysis with phenol extraction and isopropanol precipitation.
  2. Cesium chloride banding

Prepare sample according to your product type & size. Order as "BAC" or "Lambda."

Prepare sample according to your product type & size. Order as "shRNA."

Prepare sample according to your product type & size. Order as "single-stranded."