Fragment Analysis

 Amplified fragment length polymorphism (AFLP) analysis is a mapping technique that uses selective amplification to generate a set of labeled fragments of varying and characteristic sizes; also known as a "fingerprint." We have installed the GeneMapper software from Applied Biosystems for performing fragment analysis on our 48-capillary 3730xl DNA Analyzer. Multiplexed samples are simple to run: you submit the dye-labeled DNA fragments and we will give you a printout with the fragment sizes calculated. For multiples of 48, the charge is $2.60/well. For non-multiples, $3/well.

 

Sample Preparation

  • Perform labeled fragment generation using your favorite protocol (see dye set information below)
  • Clean up fragments to remove unincorporated nucleotides, primers, and salts
  • Final sample concentration must be at 100ng in 1uL. If multiplexing, samples must also be at 100ng in 1uL

 

Dye Set Information

We currently use:

  • Dye Set D-DS-30: 6-FAM (blue), HEX (green), NED (yellow), ROX (red)
  • Dye Set G5-DS-33: 6-FAM (blue), VIC (green), NED (yellow), PET (red), LIZ (orange)

We use GS500 (-250) LIZ or ROX as the size standard

You may order 6-FAM and HEX labeled oligos from us. You will need to get VIC, NED, and PET labeled oligos from Applied Biosystems.

 

Suggestions and Best Practices

  • If you are using Dye Set D, you will not be able to label your samples with ROX
  • If you are using Dye Set G5, you will not be able to label your samples with LIZ
  • Use different dye labels for PCR reactions with overlapping product sizes
  • Use a combination of dyes that display in different colors and can be detected by the same virtual filter set (Dye Set D or G5)
  • Do not multiplex primers with similar product lengths labeled with similar dyes
  • Before performing PCR, check for primer compatibility -- avoid excess regions of complementarity among the primers, choose ones with similar melting temperatures, and then test for successful co-amplification

Analysis

After samples have been run on the ABI 3730xl DNA Analyzer we will email your results. Applied Biosystems provides a free program (PC only) for analysis of their data files. We recommend this. Peak Scanner is a very simple program and we can provide directions for use. GeneMapper is available from Applied Biosystems for more convenient fragment analysis.