How to make sucrose density gradients.
We successfully prepare sucrose density gradients for ultracentrifugation without using a gradient maker. Use of gradient maker does not produce uniform gradients, ie., when made six gradients and run for polysome profiles you may not able to get uniform baselines for each gradient. Following procol is very simple and reproducible. One can make uniform (12 gradients for instance) gradients at a time.
1. Layer the dense sucrose, for instance 60 %, solution in a centrifuge tube. These tubes should be placed straght in a tube rack.
2. Overlay carefully same amount of a lighter, for instance 15 % sucrose solution onto the dense sucrose. Do not disturb the interface at this point.
3. Close the tube with a silicon rubber stopper.
4. Gently lay down the tube rack on its side, and allow to diffuse for about 1 to 3 hr.
5. Slowly straighten the tube rackt, and the gradients are now ready to use.
*This protocol was originally used in Eric Davies' lab over 20 years ago and Dr. Shunnosuke Abe and Dr. Eric Davies wrote a paper in the Memoire in College of Agriculture, Ehime University. This method really works nice. Try it!
Abe, S. and E. Davies. Quantitative analysis of polysomes using a baseline from uncentrifuged blank gradients. Memoirs of the College of Agriculture, Ehime University 31: 187-199 (1986)
A Separation of Genomic DNA Fragments
Fractionation of EcoR1 digest of pea genomic DNA using 20-40% sucrose density gradient. The fractionated DNA fragments were analyzed by an agarose gel electrophoresis and stained with Ethidium bromide.
B Separation of Polysome size classes
Polysome profile of pea stem tissue using a 15 to 60% sucrose gradient spun at 300,000xg for 45 minuts. Polysomes with various size classes after the centrifugation were analyzed using gradient fractionator with absorbance at 260 nm.
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