Automated Sequencing Services

(ABI 3100 Capillary Sequencer)

Welcome to the Laboratory for Plant Genome Technology (LPGT)

First-time users should call the LPGT manager @ 979-862-3497 to get the information of LPGT sequencing service. The LPGT's operating hours are 10:00 AM - 12:00 and 2:00 PM - 4:00 PM.

Service Fee

User-supplied PCR machine, spin columns, primers and ABI BigDye-terminator Cycle Sequencing Kit and LPGT-run : $2.50/sample. LPGT-performed sequencing reaction and run $10.00/sample

You can also use PCR machines and spin columns supplied by LPGT.

Preparation of DNA Template

DNA quality and quantity are extremely important in automated sequencing. Cesium-banding methods, alkaline lysis/PEG precipitation and QIAGEN DNA isolation procedures provide sequencing-quality DNA. We suggest that you quantify your DNA with a spectrophotometer and run it on a gel to verify concentration and purity. Resuspend DNA in water.

Suggested Purification Methods

Plasmid DNA: QIAGEN Spin Kit #27104 (50 preps) or #27106 (250) QIAGEN Plasmid Mini Kit #12123 (25 preps)

PCR Product: QIAGEN Gel Extraction Kit #28704 (50 preps); QIAquick PCR Purification Kit #28104

Cosmid, BAC DNA and genomic DNA: Consult LPGT

Template Quantity

PCR product	100-200 bp (1-3 ng), 200-500 bp (3-0 ng), 500-1000 BP (5-20 ng), 1000-2000 BP (10-40 ng), >2000 b (40-100 ng)
Single-stranded	50-100 ng
Double-stranded	200-500 ng
Cosmid, BAC	0.5-1,0 ug
Bacterial genomic DNA	2-3 µg

Preparation of Primer

Primers should be at least 18mer and desalted. Each sample will require 3.2 pmoles primer per reaction.

Calculate the Tm of your primer. One of several methods used for calculating Tm is:

Tm = [(A+T) x 2 °C] + [(G+C) x 4 °C]

This will determine the cycle sequencing program used to incorporate the fluorescent dideoxynucleotides.

Cycle Sequencing

There are several brands of fluorescent dye kits available that incorporate fluorescent dideoxynucleotides into extension products. Each kit basically comes with Taq DNA Polymerase, fluorescent-ddNTPs, & dNTPs. There are four different fluorescent dyes that distinguish between the four bases in DNA. The reaction is linear; only one primer is used per reaction. The LPGT set up the sequencing procedure based on the ABI BigDye Terminator chemistry. Refer to LPGT Cycle Sequencing Protocol site or the ABI protocol for more information on preparing reactions. The LPGT has a PE 9600 and two 9700 PCR machines available for cycle sequencing reactions. These models do not require oil and the cycle sequencing run time is about 2.5 hours. See lab technician for more information.

Extension Product Purification

Unincorporated dye terminators and residual salts must be removed from the extension products. The 3100 capillary sequencer is especially susceptible to salt in samples. The LPGT strongly recommend implementing an efficient method to remove excessive salts. This is done by preparing a commercially-available column or a LPGT spin column containing Sephadex G-50 and glass beads. Refer to LPGT Spin Column Protocol site for more information. The eluted product is dried down in a speed vac and resuspended in deionized formamide. The LPGT supplies the formamide to all individual tube users and has a Savant DNA speedvac available.

Large-scale (ABI 96 well MicroAmp plate) users can purify the extension products by isopropanol precipitation protocol. Refer to Isopropanol Protocol site.

What to Bring

Obtain a PO or RF number from your department for billing purposes. Bring purified and vaccum-dried samples to LPGT by 2:00 PM. Place in the service freezer in the labeled "Capillary Sequencer-tube" for individual tube users or "Capillary Sequencer-96 well plate" for large-scale 96 well plate users. The deionized formamide is provided for resuspending DNA samples. Covered, resuspended samples may be stored in the service -20 °C freezer up to two weeks. Fill out the "Capillary Sequencing Service Charge Form" on the desk near the sequencer area.

Automated Sequencing Runs

Automated sequencing runs are scheduled Monday through Friday with at most four plates (4 X 96 samples) per day depending on demands. Call lab personnel to check current schedule in room 139A. A plate has 95 user samples and 1 pGEM standard sample. There must be at least 15 samples on a run. If there are less, the samples will be bumped to the next run. A p-GEM standard is included to each run for instrument and run quality assurance. Failed sequences/poor data from a quality-assured run remain the user's fiscal responsibility. Samples are denatured and loaded by LPGT personnel. A laser scans the capillary and detected fluorescence is converted into actual sequence data. Run times are 50 min for a run (15 samples) and 5.0 hours for a plate (95 samples) in current 36 cm capillary setting.

Sequence Results and Analysis

Base reads of 400-600 BP are possible when samples are properly prepared and run through current 36cm capillary system. Sequence data results have a printed color electropherogram and electronic data in simple text. These results are usually available at 10:00-12:00 AM following the run . Bring a floppy or zip disk to save your files, IBM Formatted required. You can open it using PC on Mac computer operated with MacOS system. Sequence results are kept for one month.

Additional Information

QIAGEN products are available in the LPGT. See lab personnel for a product list. The ABI kits are available from Applied Biosystems (800) 327-3002 or the web http://www.appliedbiosystems.com.
LPGT personnel office hours are: 10:00 AM-12:00 PM and 2:00-4:00 PM.