Capillary (Sanger) DNA Sequencing

The GGF maintains an Applied Biosystems 3730xl 96-capillary DNA Analyzer for Image results for a SequencerSanger sequencing and fragment analysis. Users can choose to submit individual DNA samples or 96 well plates of products that are ready to be sequenced or choose to do the cycle sequencing reaction themselves and we will process the “Ready-to-Run” plates on the 3730xl. Each Sanger single tube instrument run contains a pGEM control that achieves a Q20 up to 650bp that is used to judge instrument performance. Individual customer sequence read length will vary.

Customers may download data directly from their Genesifter account. Upon request, data may be delivered via SFTP. Users are assigned password protected folders. All data resides within the PI’s folder, however independent subfolders can be obtained if requested.

 

Sample Preparation

Single tube submissions

The GGF uses BigDye version 3.1 for all sequencing.  For optimal sequencing, please follow the concentration guidelines in the table below.

To allow quality control assessments and sequencing, as well as re-runs where needed, a minimum of 10 µl of template at a concentration from the table below is required.  If primer-walking with the same template, please submit an extra 5 µl per reaction.

Samples should be diluted in high quality molecular grade water rather than TE. Quantitation is important and PCR products MUST be cleaned-up and contain only one product band. If the product does not show up as a single band on an agarose gel, it will not work for sequencing unless it is excised and purified from the gel.

Template quantity required for sequencing

Purified Plasmid or Purified PCR Product (bp)Quantity Required (ng/µl)
100-2001-3
200-5003-10
500-10005-20
1000-200010-40
>200020-50
Double-stranded Plasmid150-300
*Cosmid, BAC*0.5-1.0 µg

Plate submissions of purified DNA

We can sequence 96 well plates of purified products with one or two primers used for all of the wells (e.g., a plate of 96 plasmids sequenced with a forward or a reverse primer). Please follow the guidelines for volume and concentration of single tube samples. Please normalize your DNA template concentration before submission, we will spot check your DNA concentration to double-check normalization.

If we need to further normalize your samples a charge will be assessed to cover the cost. Note, however, that many users obtain satisfactory results by determining an average concentration of samples with a plate and using a single constant volume based on that concentration.

Plates that contain 23 or less samples will be charged as single tubes (e.g., $6 per reaction). For those plates that contain 24 or more samples (and are using the same primer set for all samples), they will be charged as if it were a full 96 well plate (UGA customers get a flat rate of $260). Thus the more templates that you put in the 96-well plate the cheaper it becomes. As this service is provided at a large discount, we need to use automated pipetting procedures. If you have less than 96 samples, please place your samples in successive columns (1,2,3).

Sequencing Primers

We have the following primers on hand that we will supply for sequencing if so desired. Please verify that the sequence of the primer listed below is the one that you need. Where custom primers are needed, a minimum of 1 µl of primer per sequencing reaction should be provided at 3.3 µM diluted in high quality molecular grade water.

Primer NameLengthSequence
M13-20 FORWARD18TGT AAA ACG ACG GCC AGT
M13-48 REVERSE24AGC GGA TAA CAA TTT CAC ACA GGA
T7 PROMOTER20TAA TAC GAC TCA CTA TAG GG
T7 TERMINATOR21TTA TGC TAG TTA TTG CTC AGC
T317AAC CCT CAC TAA AGG GA
SK17TCT AGA ACT AGT GGA TC
KS17CGA GGT CGA CGG TAT CG
SP618ATT TAG GTG ACA CTA TAG

Archiving of samples

We will be keeping samples submitted for single-tube sequencing and primers for 2 weeks to allow re-runs, if necessary.  After 2 weeks, the samples and primers will be discarded, unless prior arrangements were made to return them.  If you want the samples back please let us know at the time of submission.

For customers who do a large volume of samples with the same primers (e.g., diagnostics), we can archive those primers if requested.  For customers that are doing repeated sequencing with the same sample (e.g., primer-walking), special arrangements can be made to archive those samples for short-term experiments.

Ready-to-run sequencing

Customers who want to do the cycle sequencing themselves can submit reactions in single tubes or in 96-well plates. Reactions should be submitted wet in high quality molecular grade water (if submitted dry, please let us know how much water to rehydrate them in).  The reactions have to be in semi-skirted plates that are compatible with the 3730xl.

Submit Samples

UGA and on-campus customers:

Please go to the Genesifter page to submit your order and then bring your samples to the GGF main office (Room 161) anytime Monday-Friday between 8:00 am-5:00 pm or leave them at one of our drop-off locations:

  • Coverdell Building, Coverdell Stockroom, freezer room on first floor (Get key from Room 146).
  • Chemistry Building, CSS, ABI fridge-bottom shelf.
  • Miller Plant Science Building, Room 4602 on 4th floor.
  • Life Science Building, Genetics Department, fridge is located in their office break room B103.
  • Poultry Diagnostic and Research Center (PDRC), 953 College Station Road, room 144.

All drop-off locations have spare envelopes in which your samples and primers can be submitted. Please do not tape your samples to your submissions sheet. Because of degradation issues, RNA samples must be brought directly to the GGF main office.

Download a map of Drop off Locations (pdf)

Off-campus customers:

Please ship samples to this address:

University of Georgia
Georgia Genomics Facility
110 Riverbend Rd, Rm 161
Athens, GA 30602

Tips: Please seal plates with plate sealing foil and ship on dry-ice or in freezer packs preferably through FedEx or UPS as they deliver directly to GGF. You might also want to write “this side up” on your cooler or box.

Data Retrieval

Please go to the Genesifter page to retrieve your data.

Prices

Current Price List

Service DescriptionUGA FeeNon-UGA FeeCommercial Fee
Ready to run genotyping plate$75.00$88.50$112.50
Ready to run sequencing plate$75.00$88.50$112.50
Sequencing a 96 well plate of purified products with 1 primer$260.00$307.00$390.00
Sequencing a single tube with 1 primer$6.00$7.10$9.00
Ready to run single tube$3.30$3.90$4.95
PCR product clean-up with 1:1 SPRI; up to 100 µl sample per single tube$4.15$4.90$6.25
PCR product clean-up with 1:1 SPRI; 96-well plate (minimum 10 samples)$3.60/well$4.25/well$5.40/well
PCR product clean-up with 1:1 SPRI; 96-well plate flat fee (samples need to be submitted in a 96-well plate)$95.00$112.00$142.50
PCR product clean-up with 1:1.8 SPRI; up to 100 µl sample per single tube$5.70$6.75$8.55
PCR product clean-up with 1:1.8 SPRI; 96-well plate (minimum 10 samples)$5.55/well$6.55/well$8.35/well
PCR product clean-up with 1:1.8 SPRI; 96-well plate flat fee (samples need to be submitted in a 96-well plate)$122.00$144.00$183.00
Sephadex clean up/per plate$50.40$59.50$75.60
Sephadex clean up/per half plate$29.00$34.25$43.50
Sephadex clean up/per row of 8 wells$8.20$9.70$12.30
Formamide addition for genotyping$0.11/well$0.13/well$0.17/well
Semi-skirted 96-well plate (for 3730 sequencer)$19.00$22.45$28.50
Normalize DNA concentration in a 96 well plate$86.00$101.50$129.00
Genescan LIZ 500 or 600 Size Standard$0.80/well$0.95/well$1.20/well
GGF 500 ROX Size Standard$0.36/well$0.43/well$0.54/well
GGF 1000 ROX Size Standard$0.42/well$0.50/well$0.63/well
GGF 500 ROX Size Standard
(150 µl aliquot)
$42.00$50.60$63.00
GGF 500 ROX Size Standard
(450 µl aliquot)
$112.00$132.00$168.00
GGF 1000 ROX Size Standard
(150 µl aliquot)
$59.60$70.35$89.40
GGF 1000 ROX Size Standard
(450 µl aliquot)
$160.00$189.00$240.00

Fluorometry

Service DescriptionUGA FeeNon-UGA FeeCommercial Fee
SYBR green fluorometry$46.00 (set up fee to 35 samples)$54.30 (set up fee to 35 samples)$69.00 (set up fee to 35 samples)
+ $1.30 for each additional sample in same plate+ $1.55 for each additional sample in same plate+ $1.95 for each additional sample in same plate

Custom PCR

Service DescriptionUGA FeeNon UGA FeeCommercial Fee
PCR set up fee (max. 96 reactions)$36.00$42.50$54.00
PCR reaction charge (per reaction)$2.40$2.85$3.60

Prices are subject to change without notice. To get a  quote, please contact GGF.

Data Analysis

Any of the following programs can be used to view DNA traces, but training and support are not available through GGF .

Low-cost research data storage is available through the Georgia Advanced Computing Resource Center.

Capillary Sequencing Protocols