Molecular Ecology Notes (2004) 4, 768–771 doi: 10.1111/j.1471-8286.2004.00763.x © 2004 Blackwell Publishing Ltd Blackwell Publishing, Ltd. TECHNICAL NOTE A simple and inexpensive method for producing fluorescently labelled size standard V. VAUGHAN SYMONDS and ALAN M. LLOYD University of Texas-Austin, Department of Molecular, Cell, and Developmental Biology, Institute for Cellular and Molecular Biology, 2500 Speedway, MBB 1.448b Austin, TX 78712, USA Abstract Current methods of automated genotyping offer many advantages over traditional gel-based approaches, including reduced handling and processing times, and increased accuracy and consistency. Unfortunately, these advances have come at a substantial cost; at present, roughly one-half of the cost of automated genotyping is due to fluorescently labelled internal size standard. Here we describe detailed methodologies for generating a highly consistent, fluorescently labelled, internal size standard using polymerase chain reaction (PCR). The methods are simple and the required reagents are inexpensive, making the in-house pro- duction of fluorescently labelled size standards a more widely accessible alternative to commercially available size standards. Keywords: automated, fluorescent, genotyping, high-throughput, microsatellite, size standard Received 7 June 2004; revision received 9 July 2004; accepted 9 July 2004 Large-scale genotyping projects have become quite common, however, the per sample cost of automated genotyping remains a limiting factor for many. Roughly one-half the cost of processing DNA fragments is associated with fluore- scently labelled size standards, which have relatively few vendors. Although two recent reports (Pereira et al . 2001; Ueno et al . 2003) provide methods for producing fluorescently labelled size standards, we describe here a simpler, less expensive, and more versatile method for producing any set of size standards one may wish to use. Where other methods require multiple dye-labelled primers or elaborate schemes involving Amplified Fragment Length Polymorphism (AFLP) techniques and cloning, our method utilizes a single dye-labelled primer and PCR. For the cost of a single batch of commercially available size standard, the materials invoked here can be purchased and pro- vide enough size standard to process tens of thousands of samples. What follows is a description of the simplest version of our method for making a high-quality, cheaply amplified size standard (CASS). However, it should become obvious that the approach laid out here can be applied to any avail- able DNA template with known sequence, and many modi- fications of this protocol can be used, depending on one’s particular genotyping needs. CASS primers were designed from published sequence of the pBluescript II KS + (hereafter pBS) vector (Stratagene; GenBank Accession X52327). A single forward primer was designed from the T7 priming site with the ROX dye attached to the 5 ′ end (Perkin Elmer). From the same vector sequence, four unlabelled reverse primers were designed to yield 100, 200, 300, and 400 bp fragments when used in combination with the T7 forward primer (see Table 1 for primer sequences). Ten nanograms of pBS were used as template in four separate 20 μ L polymerase chain reactions (each with a different reverse primer), each with the fol- lowing final concentrations: 1 X supplied Taq polymerase buffer, 400 μ m (total) dNTPs, 1.5 m m MgCl 2 , 500 nm T7-ROX forward primer, 500 nm reverse primer, and one unit of Taq polymerase (Invitrogen). Standard thermal cycling conditions were used, except that a final exten- sion time of 20 min was used. This longer extension time is used to ensure that a single product of the same size is produced for each standard during PCR. Because standard Taq polymerase has inconsistent nontemplate-dependent terminal transferase activity (known as +A activity), typ- ically two products that differ by a single bp in length are generated through PCR (Clark 1988). The longer extension time drives all products to the +A form. After confirming Correspondence: Vaughan Symonds. Fax: 512-232-3432; E-mail: vsymonds@mail.utexas.edu