Biomolecular Resource Laboratory

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DNA sequencing is performed on the Applied Biosystems Model 3100 Genetic Analyzer and is offered on a fee-for-service basis. Samples submitted for sequencing and automated analysis undergo a cycle sequencing reaction that uses ABI Big Dye^TM Terminator Chemistry. The fluorescently labeled products are separated from reaction reagents and analyzed by capillary electrophoresis. The DNA sequence is automatically analyzed by the 3100 analysis software. Typical read lengths are 600 - 650 bases for high quality DNA templates and primers.

Data files are placed in individual password-protected web site folders. You can retrieve your data by logging into the lab web site http://csb.wfu.edu/brf/sequencing_data.html.

Related Sequencing Documents and Software:

• DNA Sequencing Request Form
• DNA Quantitation Using EtBr Plates
• Sequencing of PCR products
• Applied Biosystems DNA Sequencing Guide
• Sequence Scanner for Windows

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Hours of Operation/Turnaround Time

The DNA Sequencing Lab operates Monday through Friday, 8:30 a.m. - 4:30 p.m. Samples may be submitted by taking them and a completed sequencing request form (TYPED & PRINTED PLEASE) to Hanes Cold Room #5065. Please follow the instructions posted there.

Samples are delivered to the sequencing lab at the BGTC each morning. Please have your samples in the cold room before 9:00 A.M. Turnaround time for completion of analyses is usually 1-2 working days after samples are received by the lab. Data for samples received by 10:00 a.m. will be usually be available by noon the following day, depending upon sample backlog at the time.

Procedure for Sample Submission (on campus users)

Complete the sample submission form. We prefer that information on the form be typed using your computer. Save this document to your computer, complete the form and print a copy to submit with your samples.

Deliver samples to the sequencing drop off site (cold room, Hanes 5065). Place labeled sample tubes (1.5 ml or 0.5 ml Eppendorf tubes) in a plastic ziplock bag provided in the cold room. Label tubes with a number and a sample name. Please label each tube with indelible and LEGIBLE markings. Use a unique name for each sample and limit the name to no more than 12 alphanumeric characters. Please do NOT use symbols, dashes, or other non-alphanumeric characters. Number the side or top of your tube with the number corresponding to the sample request form. Be sure to write your name and the date of submission on the bag.

Place packaged samples in the sample box in the cold room, Hanes 5065. Place the completed sample summary form in the manila envelope attached to the shelves inside the cold room. A completed summary of the online sample request form must accompany your samples. Please place no more than 12 samples in each Ziplock bag.

Sample Shipping (off campus users)

Please see the instructions above for sample labeling and preparation. If you are shipping via US Mail or FedEx, place Parafilm lightly around sample tube caps. Place tubes in a small plastic bag labeled with your name, the date, and your phone number.

Please be sure to include a completed sample request form with each order.

Send samples for DNA Sequencing to:

DNA Sequencing Laboratory
Center for Structural Biology
Wake Forest University School of Medicine
Medical Center Boulevard
Winston-Salem, NC 27157
Attention: Elyse Jung     Phone 336 716-0756

Sample Preparation

The quality and quantity of your DNA are critical to achieving good sequencing results on the Model 3100 sequencer. Cycle sequencing is sensitive to impurities in the template preparation as well as to the concentration of template in the reaction mixture. Appropriate care during sample purification will lead to better results. Please consider the following guidelines when preparing your DNA sample for sequencing.

Each sample tube you submit should contain DNA and primer as follows:

	Vol	Concentration	Total mass/rxn
DNA
Plasmid (double stranded)	6 µL	100-150 ng/µL H2O *	600-840 ng
PCR Product    (200 - 500 bp)**    (501 - 1000 bp)**	6 µL	5 - 16 ng/µL H2O 17 - 42 ng/µL	10 - 8 ng 100 - 250 ng
BACs and phage DNA	12 µL	150-200 ng/µmL H2O	1-1.5 µg
PRIMER
BACs and phage DNA	4 µL	4.2 pmole/µL H2O	8.4 pmole
Plasmids and PCR products	2 µL	2.1 pmole/µL H2O***	4.2 pmole

* Use the higher concentration for large (>4 KG) plasmids.
** See Guide to Sequencing PCR products for details.
*** This is approximately 12 ng/mL for a 17 mer.

Important!!
>Do not use Tris/EDTA (TE) buffer to redissolve DNA or primers! EDTA chelates Mg++ that is required for the sequencing reactions.

Primer Considerations

Primers should meet the following criteria for successful cycle sequencing:

1. Correct concentration (see above).
2. No mismatches and no secondary hybridization sites.
3. Melting temperature of 50-55°C is best. Avoid primers with low melting temperatures (T_m <48°C). If the GC content is low, try increasing primer length to increase T_m.
4. Primer length should be 18-24 bases, preferably with a G or C at the 3' end.
5. Avoid strings of four or more of the same base (eg. AAAA or GGGG) in the primer sequence.

DNA Preparation Methods

The following methods generally produce DNA of sufficient purity for DNA sequencing. If you use an alternative method, you should determine whether the DNA preparation method is recommended for fluorescent cycle sequencing as performed in this laboratory.

Recommended methods:

1. Alkaline lysis mini-prep with a PEG precipitation. Recommended procedure is here.
2. QIAGEN plasmid preparation kits/QIAGEN Product Guide; (800) 426-8157. (800) 426-8157.
3. Wizard mini-prep/Promega; (800) 356-9526.
4. CsCl₂ purification.
5. Any commercial kits that have been tested for use with the Applied Biosystems Model 3100 DNA Sequencer.

Additional Tips:

• Completely remove traces of ethanol when using column-binding preparation methods by drying samples briefly in a vacuum centrifuge.
• Do not use more than the volume of cell culture media recommended by the method. Overloading the purification system results in DNA that is insufficiently pure for sequencing.
• Make sure the ration of A₂₆₀/A₂₈₀ for your DNA is > 1.8.
• Design PCR products to be at least 80 - 100 bp if possible.

Recommended PCR Product Purification Methods

In some cases, PCR products can be successfully sequenced after residual primers, dNTPs, etc., have been removed. Spin columns that use molecular weight cut-off filters (e.g. Amicon 100) can be used to remove low molecular weight contaminants. However, the best PCR sequences are generally those that use DNA that has been gel-purified to ensure that only one PCR product is present in the sequencing

The following vendors supply kits for PCR Product clean-up:

QIAGEN (qiagen.com)

1. QIAquick kits
2. QIAquick gel extraction kits

Promega (www1.promega.com)

1. Promega Wizard PCR Preps DNA purification
2. Promega Wizard SV Gel PCR Cleanup System

Bio 101, Inc. (www1.biogene.com)

1. GeneClean Bio101

DNA Quantitation

Although A₂₆₀ measurements can sometimes be used to accurately quantify your DNA, estimating the DNA concentration by gel electrophoresis is a more reliable method. After restriction enzyme digestion and electrophoresis of an aliquot of the DNA sample, one compares the intensity of the sample band to a band with a known amount of DNA. Plasmid DNA or digested lambda DNA (a DNA ladder) provides such a standard for a more accurate estimation of DNA concentration.

Another method involves spotting your DNA and a known amount of a control DNA on agarose plates containing ethidium bromide (EtBr). The amount of the sample DNA can be estimated visually. Click here for this procedure. We recommend that you use this method either alone or as a compliment to UV measurements.

The amount of DNA necessary for sequencing PCR products depends on the size of the product. Less DNA is required for short PCR products. Refer to the document: "Guide to Sequencing PCR Products" for more information.