Analysis of RNA splicing complexes by sucrose gradients

10 mM HEPES-KOH, pH 7.6 60 mM KC1 3 mM MgCl2 High sucrose buffer 30% sucrose (w/w) 

Gradient mixer, magnetic stirrer and pump Ultracentrifuge (Beckman) 

Precool rotor chamber and rotor to 4°C. Buckets are left on ice until use. 

The splicing reaction is prepared as described in Section 5.1.1. Store on ice before loading.8 

Sucrose gradients (5 ml per tube) are made from 2.5 ml of each of the low- and high-sucrose buffers in a 5-ml centrifuge tube according to Section 4.2.3 in Chapter 4. Store tubes on ice. 

---