Amersham hyperfilm mp

AdoMet cross-linking was performed as described^{8 (link)}. An aliquot of 2 µg of purified enzyme with 0.5 µCi of [methyl-³H]AdoMet (17.9 Ci/mmol, PerkinElmer) in 20 µl of 50 mM BisTris propane (pH 8.5), 1 mM EDTA, and 0.5 mM DTT, were transferred to a 96-well plate on ice and placed 8 cm from an inverted UV transilluminator (Compact UV Lamp, UVGL-25 (Analytik Jena US)) using UV-C (254 nm wavelength) for 1 h. The protein was then separated by SDS-PAGE, stained with Coomassie, and subjected to fluorography. The gels were incubated in water for 30 min, then incubated in 5× volume of EN3HANCE solution (Perkin Elmer) for 1 h. Gels were washed in 1% glycerol for 1 h and dried. An X-ray film (Amersham Hyperfilm MP (Cytiva)) was exposed to the dried gel for 48 h at −80 °C, followed by development of the X-ray film.

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).