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All of the Tallapoosa darter reference sequences (TDR) shown in the "Genes" section, as well as all of the PCR primer sequences that amplify the various gene segments, are available as downloadable text files. These files are grouped by the individual genes and archived as .zip files. See bottom of this page for PCR conditions. Download primers and reference sequence files for Glyceraldehyde-3-phosphate dehydrogenase: Download primers and reference sequence files for Ribosomal Protein S6: Download primers and reference sequence files for Lysophospholipase II: Download primers and reference sequence files for Phospholipase C-gamma-1: Download primers and reference sequence files for Kelch Repeat and BTB (POZ) Domain Containing Protein: Download primers and reference sequence files for the Open Reading Frame (ORF) corresponding to one exon of a gene of unknown function: PCR conditions: All of the primer pairs for all of the gene fragments utilize the same PCR conditions. For a standard 50 ul amplification: 25 ul of Qiagen
HotStarTaq Master Mix
20 ul genomic DNA (100 ng) 2.5 ul of 10 uM sense primer stock solution 2.5 ul of 10 uM antisense primer stock solution Temperature cycling program: 1) 95oC
for 15
minutes
2) 94oC for 30 seconds 3) 55oC for 1 minute 4) 72oC for 2 minutes 5) go to step 2 for 34 more cycles 6) 72oC for 10 minutes 7) 4oC - hold After PCR amplification the amplified DNA products were gel purified using the Qiagen QIAquick Gel Extraction Kit. |
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