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| DNA SEQUENCING | ||||||||||||||||||||||||||
GENOMICS PROTEOMICS BIOINFORMATICS
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DNA Sequencing Service is provided in the Core Facility using two Applied Biosystems 377XL DNA Sequencers. We sequence single or double stranded DNA either by direct sequencing as PCR products or when the DNA is cloned into a vector with a universal priming site. Custom primers can be used with both types of templates. We routinely provide 600-800 bases per reaction when the template is of high quality. The sequence reaction, using fluorescently labelled nucleotides in combination with a cycling PCR protocol, is performed by the Core Facility or, on request, by the individual researcher (pre-reacted samples). The ABI Prism BigDye Terminator and Primer Cycle Sequencing Chemistries areused for most applications. Automated DNA sequencing requires that the template be of higher purity than manual sequencing. Inadequate DNA cleanup is the most common cause of poor sequencing results. If RNA, polysacharide, protein, salts,or chemical solvents such as ethanol are present in the purified DNA sample, cycle sequencing results can be adversely affected Requirements Templates and primers should be supplied in ultrapure deionized water not in buffer. Please, supply us with 12 µl of sample per reaction (template + primer in one 0.6 ml tube). To meet the stringent requirements for successful automated cycle sequencing, templates and primers should be within a narrow range of amount and concentration. Short read lengths can result from using too much DNA template, because the dye terminators are depleted early in the sequencing reaction. Too little DNA template can result in no sequence information. Please, be conservative with your DNA estimates. For best results, better to be on the lower end of the useful concentration range. Amounts Needed for Sequencing
Samples may be dropped in the Core laboratory during regular working hours, preferably before 4 pm. Samples can also be sent by Federal Express overnight. No ice is necessary, but please seal and label your tubes. Samples should be accompanied by a completed order form which is available at the Facility or fill out the form online. The gel image of each run is saved and stored for 3 months. The results are returned electronically as files, transferred via E-mail or FTP to your University computer account or returned on a floppy disk if you provide us with a formatted disk. Color electropherograms are printed on request. Your print-outs can be picked up at the laboratory. Off-campus users will receive their results by mail. Our minimum turnaround time is 2-3 working days.
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GREENWOOD
MOLECULAR BIOLOGY FACILITY |
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1-808-956-6718 | Fax 
1-808-956-9589 | E-mail 
biotech@hawaii.edu