Procedure for requesting a PML screen. 1. Are you sure your mutant is likely to be in our library? Our mutants are selected for inclusion in our library based on their phenotype. If a mutation in your gene does not yeild one of the phenotypes we have selected for, then we are very unlikely to find a mutant for you. see the user guidelines for additional information 2. Design and test gene-specific primers to be used in the screen. See the Primer Design page for details. In our experience it is more efficient to design and test several primers at a time. We recommend designing three or four primers pointing towards the 5' end of your gene (these are your 3' Gene Specific Primers). These primers should ideally map between 800 and 1500 bp downstream of the start codon of your gene (one or more of these will be your screening primer). Combining these with two or three primers 5' primers will generally yeild at least one pair that give clean controls (see Controls). 3. Write us to obtain wildtype Mutator DNA and perform the control reactions. Several controls MUST be done prior to screening the PML library. Your paired gene-specific primers need to be tested for (i) specificity and (ii) yield. Your gene-specific primers paired with the Mu primer needs to be checked to be sure there is NO background when DNA from Mu-active maize lines is used as template.We will provide genomic DNA from Mu-active maize lines upon request. To request DNA for your control reactions, please e-mail request@chloroplast.uoregon.edu 4. Know your probe! If your probe is generated from a plasmid, you're probably pretty sure of it's identity, but if you use the gene-specific primers you designed earlier to generate the probe from genomic DNA, then we ask that you sequence the pcr product before proceeding. 5. Submit a screening request. We have an online request form you can fill out. Feel free to e-mail us at any step for clarification or advice 6. Provide control data, primers, gene map, gene sequence, and the digoxigenin-labelled probe to the PML technician in the Barkan lab. Controls:In order to evaluate your controls, and to compare to our results with your primers, we need to see them. Ideally we would like an image of the gel with the 7 control lanes on it, and an image of the blot generated by transfering that gel, and probing with your biotintylated gene-specific probe. We're quite flexable on file format, but would prefer that the image(s) have the lanes, the primer pair, and the probe clearly labled. Primers: We need 15-20 nmoles of each primer (about 110-150 µg for a 20-mer oligo). Primers can either be lyophilized or diluted in water. (The exact concentration is not critical, as long as it is clearly indicated... anywhere from 50-5000 µM.) Primers can be sent either lyophilized at room temperature or diluted and sent on WET ice via "overnight standard" mail. A copy of the information sheet from the company that synthesized the primers would also be helpful. Probes: We will need roughly 200ng of each working DIG-labelled probe. The probe can either be lyophilized and sent at room temperature, or frozen in TE or hybridization buffer and sent on WET ice via "overnight standard" mail. Please include information on how the probe was synthesized (template & primers used). The concentration and/or amount of probe should be clearly indicated. Gene Map: A simple cartoon showing the location of relevant gene features eg; start codon, stop codon and introns. The map should also indicate primer locations, and the span of the DIG-labelled probe to be used for detection of Mu insertions. Ideally we would like this map in electronic form (PICT, JPEG or TIFF are the prefered formats) Mailing address: Attn: PML Technician c/o the Barkan Lab Institute of Molecular Biology University of Oregon Eugene, OR 97403

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