Amersham typhoon phosphorimager

For northern analysis, biological triplicates were grown in parallel, aliquots of 3–5 OD were harvested, and then bulk RNA was prepared with glass beads and phenol as described [73 (link)], resolved on a 10% polyacrylamide (19:1), 7M urea, 1X TBE gel, transferred to Amersham Hybond-N+ membrane (Cytiva, Marlborough, MA cat# RPN303B), and hybridized with 5’ ³²P-labeled DNA probes (S4 Table) as described [14 (link)], followed by exposure and imaging on an Amersham Typhoon phosphorimager (Cytiva, Marlborough, MA), and quantification using Image Quant v5.2

Reactions (10 μl) were performed at 30^oC in a buffer containing: 25 mM HEPES-KOH (pH 7.6); 100 mM potassium glutamate; 10 mM Mg(OAc)₂; 1 mM DTT; 0.01 % NP-40 substitute (NP-40-S) (Roche #11754599001), 0.1 mg/ml bovine serum albumin (BSA); 0.5 nM M13mp18 ssDNA (NEB #N4040S); 30 μM dC, dG, dT, dATP; 200 μM G, C, UTP; 33 nM α-[³²P]-dCTP (Hartmann Analytic #SCP-205); 3 mM ATP; 40 nM RPA; 20 nM Pol α-primase. RPA was prebound to the ssDNA template for 10 min and then reactions were started by addition of Pol α-primase. Reactions were quenched with 10 μl 100 mM EDTA and unincorporated radiolabelled nucleotide was removed using a Microspin G-50 column (Cytiva). 1/10 volume loading buffer (10% w/v sucrose; 500 mM NaOH; ∼0.25% w/v xylene cyanol) was added to the sample before analysis on 0.7% alkaline agarose gels run in 30 mM NaOH, 2 mM EDTA for 16 hours at 25 volts. After electrophoresis gels were incubated at 4^oC in 5% trichloroacetic acid for 30 min with one buffer change after 15 min. The gel was then incubated in 500 mM Tris-Cl (pH 8) for 15 mins before being dried at 75^oC under vacuum onto Whatman 3 MM paper (Cytiva). The dried gel was imaged using a BAS-IP MS phosphor screen (Cytiva) and an Amersham Typhoon phosphorimager and on Amersham Hyperfilm MP (Cytiva).

The indicated concentration of BRCA2-derived polypeptides was incubated with 10 nM ssDNA or dsDNA in 10 μl of buffer B (35 mM Tris, pH 7.5, 1 mM MgCl₂, 1 mM DTT, 100 μg/ml BSA, and 50 mM KCl) at 25 °C for 5 min. Reactions were resolved by native gel electrophoresis in 8% polyacrylamide gels and TAE buffer (50 mM Tris-acetate, pH 7.5, and 0.5 mM EDTA) at 50 mA for 1 h. For detection of ³²P-labeled DNA, gels were dried on a sheet of DEAE paper or Hybond-N (Amersham) and analyzed in Personal Imager FX (Bio-Rad) using ImageLab 5.2.1(Bio-Rad) or Amersham Typhoon phosphorimager (Cytiva) using the ImageQuant 8.2 software (Cytiva). Gels containing Cy-5 labeled DNA were analyzed using a ChemiDoc imaging system and the ImageLab software 5.2. (Bio-Rad). Mean values and standard deviations were calculated and presented using GraphPad Prism 8.4 or Microsoft Excel 16.