Figure 4. SUCROSE GRADIENT ZONAL CENTRIFUGATION
The H-CMP-labeled product was isolated from the scaled up RNA polymerase reaction mixture using
carrier EL coli RNA. The tritium-labeled materials (200 pi) obtained from the nucleic acid
extraction of the scaled up reaction mixture were layered over 5 ml sucrose (Schwarz Mann special
density grade) gradients, 5 20% w/v, containing 0.01M Tris-HCl pH 7.6 and 0.02 M NaCl and centri
fuged (SW 39 rotor) 6 hr at 4 C. The E_. coli RNA standard was centrifuged in a separate tube. The
gradient was analyzed by pumping the gradient through the Gilford J cell, delivering the densest
portion first with a bottom probe. The ultraviolet absorbancy was monitored with the Gilford re
corder. Fractions were collected and measured for radioactivity by placing 200 pi samples in Brays
scintillation fluid and counted in the scintillation counter. The ultra-violet absorbancy profiles
for the products ( ) and _E. coli RNA ( ) and the radioactivity profile ('*) are shown