4.3. Cloning of RT-PCR Products Corresponding to AR Variants

Primers (forward primer: 5′-CTTAGGATCCATGGAAGTGCAGTTAGGGCT-3′ and reverse primer: 5′-AAATGCGGCCGCTCACTGGGTGTGGAAATAG-3′) were designed to amplify the full-length coding region of the AR gene from cDNA samples of VCaP, LNCaP, or MDA-MB-453 cells. The initiation (ATG) and stop (TCA) codons of the AR gene in the forward and reverse primers respectively are in italics and BamHI and XhoI sites (underlined) used for cloning in the forward and reverse primers respectively are underlined. PCR was conducted by an iCycler (Bio-Rad, San Diego, CA, USA) in 50 µL of 1× Phusion GC buffer containing 1 unit of Phusion^® High-Fidelity DNA polymerase (Thermo Scientific, Scoresby, VIC, Australia) and 100 ng of cDNA from VCaP, LNCaP, or MDA-MB-453 cells. Resultant PCR products were then cloned into the TA vector (pCR^®2.1, Invitrogen, Scoresby, VIC, Australia). Inserts were sequenced bidirectionally using primers T7 and Sp6 by the Flinders Sequencing Facility (Adelaide, SA, Australia).