Protocol Online logo
Top : Forum Archives: : General Lab Techniques

CsCl ultracentrifugation - No satellite band (Jun/20/2008 )

I scaled down a 4mL protocol to a 2mL one. 300ug of DNA; 20uL EthBr; 62uL ActinoM-D; CsCl added until refractive index of 1.390-1.400; to 2mL with water. 40,000 rpm for 48 hours and no middle band. Any hints?


Probably you thought about this, but did you use a different centrifuge? Did you calculate RCF? RPM is not a very good measure of centrifugal force... Other idea: did you add less DNA? Could be invisible, because of the smaller amount. And yet another: The centrifugal force is higher towards the lower end of the tube, so the force distribution may be different when you cf a smaller amount of solution in the same kind of tube. That can cause a different density distribution within the gradient, and you DNA can be somewhere else, not in the middle. Can be very close to the bottom.


What if I tried a slower spin speed?