Computational protocol: Targeted identification of genomic regions using TAGdb

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[…] A 4.5 Kbp fragment of the Arabidopsis genomic region beginning 1000 bp upstream of the transducin/WD-40 repeat family gene AT3G51930 (referred to as AtWD40) was used to query all currently available TAGdb datasets (Table ). PCR primer pairs were designed from Brassica rapa tags aligning to the query sequence for the amplification of the WD40 genomic region in Brassica rapa cv. Chiifu (Table ). PCR products (a) and (b) were amplified from 60 ng of Brassica rapa cv. Chiifu DNA using 0.5 μM forward and reverse oligonucleotide primer, 1 U of Phusion Hot Start High-Fidelity DNA Polymerase (Finnzymes), 1× Phusion GC Buffer (Finnzymes) with 1.5 mM MgCl2, and 200 μM each dNTP in a PTC-200 Thermocycler (MJ Research). Cycling conditions were 98°C for 30 sec followed by 35 cycles of 98°C for 10 s, 63°C for 30 s and 72°C for 1 min, with a final extension of 72°C for 10 min. Amplified products were visualised under UV light on 1% TAE-agarose gels containing Ethidium Bromide and using the GeneRuler™ 1 Kb marker as a size standard. PCR products (a) and (b) were cloned using the pGEM®-T-easy (Promega) and pCR®-XL-Topo® (Invitrogen) vector systems respectively and sequenced using T7 and SP6 or M13R primers, and the internal primers 1F, 2R, 3F, 3R, 4F (Table ) and WD40_3pseqR (5'-TGGAAGAGATTAGGTGAAATGTGA-3'). A consensus sequence for the BrWD40 region (3985 bp) was generated in Geneious Pro [] from a contig assembly of sequenced products. Alignments and dotplots (window size 10, threshold 35) were generated in Geneious Pro using MUSCLE [] and ClustalW [] with default settings. […]

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