University of Pennsylvania
DNA Sequencing Facility

Genotyping⁄Fragment sizing

ABI 3130XL DNA Analyzer

Genotyping/Fragment sizing is currently done on ABI (Applied Biosystems) 3130XL capillary sequencer, for microsatellite-based genotyping used in linkage analysis, identifying VNTR alleles in a genetic screen, mapping genomic clones, or any other purposes. For this PCR products need to be generated with one primer labeled on the 5′ side preferably with an ABI synthesized dye FAM, HEX (or VIC), NED etc (see below). Submit about 3-4 μl PCR products (single or pooled) in 0.2 μl strip tubes or in a reaction plate. Samples can also be submitted in a ready-to-load format (PCR products + ROX size standard + formamide already added) on an ABI 96-well reaction plate.

Dye Combinations:

  • DS 30: 6-FAM, HEX, NED & ROX
  • DS 31: 6-FAM, VIC, NED & ROX
  • DS 32: 5-FAM, JOE, NED & ROX
  • DS 33: 6-FAM, VIC, NED, PET & LIZ

Analysis for sizing and allele calling is done with ABI GeneMapper (Windows) software only. Analysis of Mac converted raw data with Mac software does not work any more. Genotyped data in excel is put in the user folder on the server or e-mailed. Users are encouraged to check the raw data at the facility. Training on how to use GeneMapper is provided at no extra charge.

SNaPshot Multiplex system

SNaPshot

Developed by Applied Biosystems this is a primer extension based method that enables multiplexing up to 10 SNPs. The applications include screening and confirmation of multiple SNPs (up to 10) regardless of chromosome positions with a small amount of amplified templates, detection of DNA methylation at CpG sites and fingerprinting BACs. Please contact the Facility for further details.

Looking at the Data

Applied Biosystems offers a free downloadable, Peak Scanner™ Software v1.0 for DNA fragment analysis. Peak Scanner software separates DNA fragments by size, provides a profile of the separation, and precisely calculates each fragment size. The software allows one to view, edit, analyze, print, and export fragment analysis data generated on all Applied Biosystems sequencers.

Genotyping Request Form

The DNA Sequencing Facility is an Abramson Cancer Center Shared Resource that is approved and partially funded by the National Cancer Institute.

abramson-hup

Announcements:
Collection boxes have now been put in the lobby of BRB next to the UPS box and the 1st floor lobby of TRC (near the FEDEX box at the back).  Samples for DNA (Sanger) sequencing can be dropped off into the boxes.  Submission instructions and more information here.


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