Genomics/Microarray Facility

Services (DNA Sequencing)

Contact: Ms. Meifang Xie
Tel (215) 898-3991
Fax (215) 898-4521

MeiFang Xie has been running the sequencing facility since 1995. The facility uses a ABI 3100 capillary machine for Sanger-type sequencing with fluorescently-labeled dye-terminators, the ABI four-color detection, and sequence analysis system. The ABI 3100 is a 16 capillary machine that can sequence 600-650 clean basepairs for over 100 sequences in a 24 hour period. The 3100 can also be configured for fluorescent fragment detection and microsatellite analysis and is equipped with Gene Scanning Software.

How to use the facility

Click here for the DNA Sequencing Submission Form

Purified DNA is provided by the investigator with an order form specifying the quantity, form (double-stranded, single-stranded, or PCR product) and size of the DNA. Ms.Meifang Xie is experienced with sequencing PCR products and can assist in the estimation of PCR product DNA quantity from ethidium-bromide-stained gel pictures if necessary. It is essential that the size and concentration of PCR products are determined accurately prior to sequencing. Lambda or Bac clones require submission of greater quantities of DNA. Click here for pricing information.

Procedures
For each sequencing run, investigators must provide, in numbered, 1.5 ml Eppendorf tubes.:

• (A) double-stranded DNA: 1-1.5 µg of purified DNA at 0.2- 0.25 µg/µl
• (B) single-stranded DNA : 1 µg of purified DNA at 0.1- 0.125 µg/µl
• (C) PCR products: at least 15ng of purified DNA per 100 bp of PCR product in 5ul water.
• (D) Lambda phage or Bac clones: 10ug purified DNA at 2-2.5 µg/µl

A name designation of 8 or fewer characters, and the primers to be used for each run must be clearly indicated on the submission form.

• M13 forward, M13 reverse, T7, T3, and SP6 primers are provided by the facility at no additional cost. Specific primers must be supplied by the investigator at 3.2 pmoles/µl.
• Depending on well availability, DNA samples delivered by 10:00 am will be available the next day at 3:00 pm. DNA samples for which the dye-terminator reactions have been run by investigator can be delivered before 2:00 pm for pick up the following day at 2:00 pm. No sequencing reactions are run on Fridays.

What to expect from the facility

• For clean double-stranded templates, extremely accurate sequence reads of 600-700 bases are obtained routinely.

• sequence traces are edited and text-files of sequence data are e-mailed

• the original result folder from the ABI basecaller is placed in a computer folder for electronic retrieval if needed.
• other modes of delivery can be arranged.

Special Considerations*

*outside users must provide a PO# with submission form or establish a Wistar account before sending samples.

***A rate of $5 per lane is charged for Wistar staff wishing to run their own dye terminator or dye primer reactions and have the facility sequence the DNA. Samples must be delivered as dried precipitates and will be resuspended with formamide:EDTA prior to loading, free of charge.