Strip Chart Counting of 32P in
Gel Electrophoresis Strips
Electrophoresis on polyacrylamide gels is widely used for the fractionation of ""P-labeled nucleic acids (1). Isolation of the compounds so separated usually involves localization by autoradiography followed by excision and extraction of the relevant gel segments. Since extraction yields are variable (2,3), quantitation of 3"P-distribution in the gel patterns requires an additional step, such as slicing and Cerenkov counting or densitometry of X-ray films (4).
As an alternative, we propose that "2P-labeled gels may be scanned by strip chart counting (SCC) in a gas flow counter. Being rapid as well as non-destructive, this procedure is useful for the characterization of large numbers of gel patterns.
32P-labeled RNA of the E. co/i phage MS2 was isolated as described by Glitz (5) and contained 0.51 X 10 cpm/pg of RNA, determined by Cerenkov counting in H,O at about 50% efficiency. T, nuclease of Aspergillus oryzue (6) was purified from dried mycelia (Sankyo, Tokyo, Japan) as described (7). Slab-type electrophoresis was carried out in an EC 490 cell (E.C. Apparatus Corp., St. Petersburg, FL) with a Teflon slotformer (8,9) producing sample wells of 1 mm thickness and 2 cm width. After electrophoresis, the slab (0.3 x 20 X 40 cm) was briefly rinsed in water, and placed on a sheet of plastic film (Saran wrap). Gel strips were cut out using the sample well and bromphenol blue marker position as guides, and sealed in a single thickness of Saran wrap. For SCC, the gels were counted in an Actigraph III thin layer plate conveyor system with a micromil window (Model 1006) connected to a Model 8703 decade scaler and printer (Nuclear Chicago, Des Plaines, IL) operating at 1050 volts, time constant 10 s, 1000 cpm scale, at a chart speed of 60 cm/h. Total counts were obtained by summation of scaler counts set to a 0.5 min time interval. The background correction (blank gels) was 6 cpm. During SCC, the detector block was mounted on top of the conveyor system providing 2~ geometry. To decrease stray radiation, two collimator slits were used, the lower one (0.75 mm x 3.8 cm) situated about 1 mm above the gel strip and centered 5 mm below the upper slit (1.5 mm x 3.8 cm). For scintillation counting. the relevant gel areas were excised in 1 mm slices, and counted in 10 ml of H,O by Cerenkov counting with a O-1000 window setting. The background correction was 43 cpm.