I want to study rRNAs in polysomes from oocytes. I will use RT-PCR and then gene-specific PCR to identify rRNA. I have following questions.
1) Is sucrose gradient for polysome purification is necessary or I can just use sucrose cushion?
2) If sucrose gradient is important, could you offer me the easiest protocol how to make sucrose gradient?
3) In several protocols I've seen people have used SW41 rotor for gradient centrifugation, but we don't have this rotor. Can I use mls50 rotor (5 ml tube) or it is too small volume for gradient?
If anyone has any experience for polysome purification for rRNA detection I would appreciate the help.
Thank you,
Asya
A sucrose gradient is necessary. With a sucrose cushion it is all or nothing. The gradient allows for different layers to be taken and to then select based on the appropriate weight/size.
Here is a protocol for this:
https://drummondlab.org/protocols/protocol/sucrose-gradient
Any volume should work for the gradient it just may require some extra testing and setup experiments.
Joshua Beytebiere Thank you! Unfortunately, we don't have a gradient maker and a fractionator.
Could you give some advice, please, how I can do this manually?
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