Core Facility (Houghton'04)

DNA Sequencing

Contact: Ping Jiang
Rm: NSC 448
Tel: (404) 413-5370

Software!!!
Software to view chromatogram.

DNA Sequencing Service Request Forms
DNA Sequencing Sample Form
DNA Sequencing Premix Form

Charges :	$10.00/sequencing rxn. for Plasmid DNA and PCR products
	$15.00/sequencing rxn for BAC DNA, Cosmid DNA, Genomic DNA

Two 3100 Genetic Analyzers (Applied Biosystems) are the instrumental backbone of the DNA Sequencing facility, with each providing an automated capillary electrophoresis system that can separate, detect and analyze up to 16 capillaries of fluorescently labeled DNA fragments in 1 run. The 3100 Genetic analyzer analyzes DNA using sequencing and fragment analysis.

Website with different applications, frequently asked questions and protocols:
ABI Documents and Brochures about DNA sequencing

DNA Sequencing Service Request Form

Sample Prep:
PCR Prep/Cleanup
Basic Sample Prep
Template Preparation guidelines
Premix Sample Prep
DS plasmid DNA
PCR Product

Support:
Primer Selection guidelines
Core Facility Primers in stock
Troubleshooting

Software!!!
Software to view chromatogram. Several choices are available through Chromas, Four Peaks (Mac) and LaserGene (PC and MAC).
Chromas Lite FREE - view and print chromatogram, export sequence in text or FASTA format, reverse sequence and search for exact matches or redundent codes.
Chromas 2.31 60 day trial; $65 single user - same as Chromas Lite plus export in batches and additional formats, search for sequence matches or optimal alignment, display translations in 3 frames along with the sequence, copy and paste image into other documents.
Chromas Pro 60 day trial; $225 single user - same as Chromas 2.31 plus Map open reading frames and translate OFRs instantly, BLAST searches through NCBI, multiple alignments by interfacing with ClustalW, Display translations when editing nucleotide sequences, perform reverse translations and plot nucleotide degeneracy and more.
LaserGene (Mac OSX and PC) 30 day trial; Brinton lab for details - SeqBuilder allows you to edit, map and do virtual cloning; EditSeq allows you to translate DNA, BLAST searches, ORF identification and selection . There are addition modules that can be purchased.
Four Peaks (Mac OSX) FREE - Edit DNA sequence and translation, reverse sequence, change translation frame, BLAST query directly.
NEW!!!

Sample Prep

PCR Cleanup
QIAquick Spin Handbook

Basic Sample Prep
TEMPLATE RECOMMENDATIONS FOR DNA SEQUENCING:
Template preparation guidelines:
1. Concentration and amount of template
DS plasmid DNA 100-300 ng/μl; 1 μg/direction
SS or phagemid DNA 50-100 ng/μl; 500 ng/direction
PCR fragment 10-20 ng/μl for every 200 bases of length. (25-50 for every 500bp PCR length, etc)
Please always give sufficient DNA/primer for two sequencing reactions. This allows us to repeat a sample -in case of technical failure- without having to contact you for more sample.
2. For plasmid template preparation, we recommend using Qiagen QiaPrep plasmid kit. Both give you the best results. In addition to Qiagen procedures, CsCl DNA preps and the Promega Wizard DNA purification system yield high quality template DNA.
3. Concentration of template should be based upon OD260 and verified by agarose gel electrophoresis.
4. Template should be in sterile distilled H2O (preferred) or TE (10mM tris, pH 8.0, 0.1mM EDTA).
PCR template
1. an aliquot of the product should be run on an agarose gel, along with a molecular weight marker.
2. If you only see one sharp band, the product does not need to be gel purified.
3. If you see more than one band, the material should be extracted and purified from an agarose gel, using the QIAquick Gel Extraction kit. This process removes the excess primers and nucleotides from the PCR reaction, and is essential to obtaining clean sequences. A significant improvement is seen when three washes are performed.
4. If the products do not require gel extraction, they should still be put through QIAquick Gel Extraction kit. This process removes the excess primers and nucleotides from the PCR reaction, and is essential to obtaining clean sequences. A significant improvement is seen when three washes are performed.

Premixed DNA-primer guidelines
Please follow the guidelines to provide template and primer premixed in one tube:

For DS plasmid DNA:
λ 600-800 ng ds plasmid DNA template
λ 2 µl 4 µM primer
λ x µl sterile water
λ 28 µl total volume

For PCR Product:
λ 10-20 ng templates for every 200 bp of PCR fragment length.
λ 2 µl 4uM primer
λ x µl sterile water
λ 28 µl total volume
(Mix 25-50 ng for every 500 bp PCR fragment)

This total volume is sufficient for two sequencing reactions which allow us to repeat a sample in case of technical problems.

Support
Primer Selection Guidelines:
1. 18-28 nucleotides in length.
2. 50% G/C content.
3. G & C “clamps” on the 3’ and 5’ ends (at least a single G or C residue)
4. Primer should be at least 20-30 bases long at 5’ of region to be sequenced.
5. Avoid multiple Thymidine residues on 3’ and 5’ ends.
6. Avoid primers with long runs (more than 3 or 4) of a single base.
7. Avoid primers with tendency to form strong intramolecular base pairs or primer primer dimers.
8. Melting temperature 55-65??.
9. Check primers for specificity in annealing to template. If possible use a computer program to design primers.
10. PRIMERS SHOULD BE 4μM in CONCENTRATION (For average 20 mer, 4μM corresponds to approximately 27 ng/μl) and at least 15μl.

Primer Stocks:
We have the following primers in stock: T7, T3, SK, KS, M13-21, M13REV, PCRIIT7, PCRIISP6, SP6, pcDNA3.1 REV, and T7 terminator.

Troubleshooting:
1. Noisy sequence
a. carry-over of primers used in the PCR. This is remedied by passing the template through the QIAquick Gel Extraction kit, again. If sequencing is performed with an internal primer, then the presence of other primers from the PCR is apparent because the major sequence data terminates and is followed by a lower level of sequence coming from priming with the contaminating outer primers.
b. the presence of two PCR products in the template, both of which sequence with the sequencing primers. Although the efficiency of sequencing of the contaminating band may be lower than that of the desired product, it may add noise at a detectable level. The presence of a second PCR product may not be observable from standard agarose gels, but can be diagnosed from acrylamide gels or by running on “PCR Purity plus Gels”.
c. Concentration of PCR product too high. Repeat sequencing with one-half or one third the DNA and compare noise level to first run. Brightness of bands on the fluorescent gel image should be a first indicator of over-load.
2. Weak signal
a. Usually means there isn’t enough template for sequencing. Check gel picture.
b. Due to improper concentration of sequencing primer. Check O.D of primer

This information is given as a guide to the facilities and instrumentation available in the DNA/Protein Core facility at Georgia State University. If you have any concerns or thoughts about the content of this website please contact: John Houghton (404) 651- 0549: jhoughton@gsu.edu)