TITLE: University of Helsinki and Natural Resources Institute Finland (Luke) protocol for DNA extraction and multiplex PCR genotyping of 17 microsatellites for pikeperch (Sander lucioperca L.).
AUTHORS: Jarmo Koskiniemi, Marja-Liisa Koljonen, Tuomas Leinonen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this protocol we describe laboratory methods for DNA extraction and multiplex genotyping of pikeperch with microsatellite markers. The protocol has been used in several studies at the University of Helsinki and the Natural Resources Institute Finland (Luke). Publications from these studies are listed in the attachment.</div></div>

[STEPS]
?. DNA is extracted from dried scales or from fins or other tissues preserved in alcohol, frozen or fresh. The extractions are done using Qiagen DNeasy or DNAeasy 96 Blood & Tissue Kits with the kit manual’s ‘Animal Tissues’ protocols with a few modifications for the egg samples.
?. Usually only 1 scale, or if they are very small, 2-3 scales are used. From the tissue samples, a small piece (max. 10 mg) is cut and the pieces from samples in alcohol are kept overnight in open tubes to let the alcohol evaporate.
?. The PCRs are done using Qiagen Type-it Microsatellite Kit. The kit manual’s ‘Optimized cycling protocol for multiplex PCR amplification of microsatellites’ is used with the annealing temperature of 56°C, but with modifications on the reaction volumes. When the samples are fresh, or have been kept frozen or in alcohol for max. 1 year, 10 ul reactions are used. For max. 1 year old dried scales, 15 ul reactions are used. If samples are kept frozen or in alcohol for more than 1 year or if the dried scales are older than 1 year, 25 ul reactions are used. The extracted DNA is usually used without dilution. When the samples are very old, the extracted DNA is concentrated to 1/10 of the original volume by keeping the DNA in open tubes at room temperature. For the 10 ul reaction, 5 ul of kit's master mix and 3 ul of extracted DNA are used. For the 15 ul reaction, these volumes are multiplied by 1.5, and for 25 ul reactions by 2.5.
?. Microsatellite genotypes are detected with an Applied Biosystems ABI 3130 automated DNA sequencer, and analysed with GeneMapper analysis software v5.0, with the size standard of Applied Biosystems GeneScan 500LIZ. Automatic outputs are checked for errors and corrected manually.
?. 17 microsatellite loci are analyzed in two multiplex-reactions. The multiplexes, primer sequences, primer concentrations, dyes, loci names in references and GenBank, references for each locus and GenBank accession numbers are:  Locus	    Multiplex	    Forward primer sequence (5'-3')	    Reverse primer sequence (5'-3') MSL_1	    MP2	            TGTTTGTCAGCGTCAAGAGG	    TTCCGCTCCAACATATCACA MSL_2        MP2	            TTTTCACACCGTGCATGACT	            ACCCTCAGCCTCTGTGTACG MSL_3        MP2	            CCGGCATCCATACACCTTAC	            CACACCTGTGTCTGCCTAACA MSL_4	    MP2	            TCAAGACCCCAGAACCAATC	    CAGACAGCTAAGAGAACAACAGG MSL_6        MP2	            GTCGTCATCGTCAGCACAGT	    ACTACACGGGACGCTGGA MSL_7	    MP2	            CACACAGCAGCATGTGACAA	    GGCACGGAGGTAGAATGGTA MSL_8	    MP2	            AACACCTTCCTTCGTCCATC	            CGTGTTTGCCTCACACAAAG MSL_9	    MP2	            GCATCACTTGCGTCACTTTC	            GCAGTCAGTGCTTGAAGTGG Pfla_L3	    MP1	            GCCGAATGTGATTGAATG	            CGCTAAAGCCAACTTAATG Pfla_L8	    MP1	            GCCTTATTGTGTGACTTATCG	    GGATCTTTCACTTTTTCTTTCAG Pfla_L9	    MP1	            GTTAGTGTGAAAGAAGCATCTGC	    TGGGAAATGTGGTCAGCGGC Svi_18	    MP1	            GATCTGTAAACTCCAGCGTG	            CTTAAGCTGCTCAGCATCCAGG Svi_33	    MP1	            CAGGACTGCTGTGTATAGACTTG	    GATATAGCTTTCTGCTGGGGTC Svi_4	    MP1	            ACAAATGCGGGCTGCTGTTC	    GATCGCGGCACAGATGTATTG Svi_6	    MP1	            CATATTATGTAGAGTGCAGACCC	    TGAGCTTCACCTCATATTCC Svi_L7	    MP1	            GATGTGCATACATTTACTCC	            GCTTTAATCTGCTGAGAAC Svi_L8	    MP1	            GCTTATACGTCGTTCTTATG	            ATGGAGAAGCAAGTTGAG  Locus	    Primer concentration (µM)	    Dye	    Orig. locus name	Reference 	   GenBank Accession no. MSL_1	    0,20	                                            VIC	     MSL-1	                R1	                    EF694018.1 MSL_2	    0,10	                                            NED	    MSL-2	                        R1	                    EF694019.1 MSL_3	    0,05	                                            NED	    MSL-3	                        R1	                    EF694020.1 MSL_4	    0,10	                                            6FAM	    MSL-4	                        R1	                    EF694021.1 MSL_6	    0,10	                                            PET	    MSL-6	                        R1	                    EF694023.1 MSL_7	    0,05	                                            VIC	    MSL-7	                        R1	                    EF694024.1 MSL_8	    0,05	                                            VIC	    MSL-8	                        R1	                    EF694025.1 MSL_9	    0,20	                                            6FAM	    MSL-9	                        R1	                    EF694026.1 Pfla_L3	    0,40	                                            6FAM	    Pfla L3	                R2	                    AF211828.1 Pfla_L8	    1,00	                                            PET	    Pfla L8	                R2	                    AF211833.1 Pfla_L9	    0,10	                                            NED	    Pfla L9	                R2	                    AF211834.1 Svi_18	    0,20	                                            6FAM	    Svi18	                        R3	                    G36964.1 Svi_33	    0,05	                                            6FAM	    Svi33	                        R3	                    G36967.1 Svi_4	    0,10	                                            NED	    Svi4	                        R3	                    G36961.1 Svi_6	    0,10	                                            VIC	    Svi6	                        R3	                    G36962.1 Svi_L7	    0,20	                                            VIC	     Svi L7	                R3	                    AF144740.1 Svi_L8	    1,00	                                            PET	     Svi L8	                R3	                    AF144741.1  R1: Kohlmann K, Kersten P  (2008). Isolation and characterization of nine microsatellite loci from the pike-perch, Sander lucioperca (Linnaeus, 1758). Molecular Ecology Resources 8:1085-1087. R2: Borer SO, Miller LM, Kapuscinski  AR (1999). Microsatellites in walleye Stizostedion vitreum. Molecular Ecology 8:336-338. R3: Wirth T, Saint-Laurent R, Bernatchez L (1999). Isolation and characterization of microsatellite loci in the walleye (Stizostedion vitreum), and cross-species amplification within the family Percidae. Molecular Ecology 8:1960-1962.