We are trying to prepare continuous/discontinuous sucrose gradient for isolating chloroplast membranes. Can anyone suggest which is the best gradient maker (which company) or can we prepare the gradient manually?
I used to use a Beckman gradient maker with piston drives for two syringes as sucrose gets pretty viscous at high concentrations. Before that I used to make linear gradients by hand and verify it by measuring refractive index. That was equally good. The machine would be better by log gradients if I remember correctly.
Thank you sir, could you please tell me how do u measure refractive index? I have prepared discontinuous gradient manually but still not sure if same can be done with continuous gradient also.
Thanks
You use an Abbe's refractometer. If you make a discontinuous gradient of sucrose, say with steps of 0.2 M and leave it aside in ice(Make sure it does not sink or wobble) for a couple of hours it becomes continuous. That is why you check with a refractometer. You can even estimate sucrose, more tedious.
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