I have a protocol for digesting yeast chromatin with MNase and when I run the DNA on a 1.5% agarose gel, the majority of my species are indeed in the mononucleosome form. However, I wish to do an IP with the mononucleosomes so even the smallest contamination with di and tri-nucleosomes is a problem.
I have found a few protocols that suggest using a 5-29% sucrose gradient. I have tried this but have not detected the peaks I am interested in (everything was at the top of my gradient).
So my first question would be: has anyone made such sucrose gradients and what is the best way to make them? Overnight at RT? I used a special machine that makes them in 30 seconds.....but can I trust it?
I'm wondering whether my experiment did not work because the gradient was not made properly.
Thank you for your help.
Hi Julia,
Ultracentrifugation in sucrose gradient is a challenging procedure and I would not expect it to work for anyone for the very first time. The following parameters are of importance:
1) the gradient quality. The gradient can be prepared by either:
- layering several sucrose concentrations on top of each other very carefully in a 5 or 50 ml disposable tubes that fit the swinging bucket rotor tubes and let stay overnight to smooth gradient;
- using programmed pumps that will mix the gradient in a proper ratio.
In both cases, once you prepared the gradient be very careful not to shake or vibrate the tubes to avoid gradient distortion. The same rule is applied once the run is over and you are about to collect the separated fractions.
2) The initial amount and quality of the material to be separated: should be not to much but also not to little (unless it is radioactive and you can easily detect it ).
Too much material will not be fractionated properly, while to little may result in difficult collection and detection.
3) Collection of the material once the run is over. It is usually done by drop collection method: seal the top of the tube with parafilm, puncture the tube on a side near the bottom with a needle and collect fraction by drops (10-15/fraction if it is 5 ml and 1-1.5 ml if it is 50 ml volume). Do not squeeze or shake the tube, otherwise the gradient and fractionated material will be disturbed and cross-contaminated.
4) Finally, in some cases, it is necessary to concentrate the fractionated material prior the analysis (under either native or denaturating conditions).
Good luck
"Everything was at the top of my gradient" suggests the centrifugation conditions were not right.
I would suggest to check the centrifugation conditions.
Thank you for the answers guys. I'm inclined to suspect there was a problem with the gradient per se because I followed a published protocol. I centrifuged at 36000 rpm for 16 hours......
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