Kapa dna polymerase

Three days post transduction, genomic DNA was isolated using QuickExtract (catalog #QE09050, Epicenter) according to the manufacturer’s instructions. PCR amplification of the genomic DNA was performed using 700 ng genomic DNA and specifically designed forward and reverse primers (Supplementary Table 3) and KAPA DNA polymerase (KAPA Biosystems). The PCR product was purified and 315 ng of the purified DNA was denatured and reannealed in the presence of NEB restriction enzyme buffer 2 in a thermocycler. The reannealed DNA was mixed with enhancer and Surveyor nuclease (IDT) according to the manufacturer’s instructions and incubated at 42 °C for 50 min before separation on 4–20% Tris/Borate/EDTA gels. The DNA bands were visualized using SYBR gold stain (Thermo Scientific).

To generate the yeast two-hybrid plasmids, full-length (1–1143) or C-term (620–1143) was inserted into pGBKT7 (Clontech) using SmaI/SalI restrictions sites. The BRCv (687FR→AA) mutation was created by Quikchange mutagenesis (Agilent Genomics) using Kapa DNA polymerase (Kapa Biosystems) and screened with novel silent restriction sites. RAD51 was PCR amplified from pGAT3-RAD51 (81 (link)) with primers that included MfeI and XhoI sites and then ligated into pGADT7 (Clontech) digested with EcoRI/XhoI. The yeast strain SFY526 (MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, canr, gal4-542, gal80–538, URA::GAL1UAS-GAL1TATA-lacZ) was transformed with the appropriate plasmids (see figure legends) according to the manufacturer's instructions using the lithium acetate procedure (Clontech Matchmaker 2 manual). Liquid cultures were grown overnight in standard dropout (SD) media lacking tryptophan and leucine. The cells were restreaked onto SD/-Trp/-Leu and incubated at 30 °C for 2 to 3 days before performing a colony-lift β-galactosidase assay (X-gal, Sigma-Aldrich) according to the Yeast Protocols Handbook (PT3024-1, Clontech).