I tried to dissolve sucrose to make 3.7 M, but I could not fully...

Hi Hyong Woo,

sorry I just realized you asked for some extra information in your last request!

About the pellet size: the 50 uL gray pellet is normal with such amount of starting material. Starting with large amounts give you the possibility of try more conditions (temperatures, times, MNase concentration) but gives you a less pure pellet. Lately I am performing nuclei purification with less starting material and I am getting cleaner extractions. I also try to do a mild grinding that although decreases the efficiency but increases the purity and the integrity of the nuclei extracted.

About the NanoDrop mesurements: have you tried diluting the sample >1/20? Higher dilution should decrease the interference of cell debris and other substances. One alternative to the NanoDrop is to run an agarose gel and quantify the bands before the MNase treatment.

Hope this late answer still helps,
Sincerely, Jordi

Hi Hyong Woo, Thank you for pointing out. The concentration of sucrose in Nuclei extraction buffer C was changed from 3.7 M to 1.7 M as requested by Dr. Jordi Moreno-Romero.

Good Luck,
Bio-protocol Editorial team

Hi Jordi~! Thank you very much for your confirmation. I just finished pilot experiment to see how this protocol works.

From 2 grams of Arabidopsis leaf sample, I was able to get a big white- or grey-colored pellets, which seems like a nuclei, after the sucrose gradient centrifugation (Step 9). Nuclear pellet looks to me more than 50 uL in volume.

Do you usually can get such a large amount of nuclei pellet from 2 g Arabidopsis leaves?

And, more importantly, I was not able to measure the DNA concentration with the nanodrop at OD260 (I used 1/20 diluted samples in distilled water). It seems like to be due to the interference of white colored floating matters... (lipids???)

Could you provide a more detailed procedure how to measure DNA concentrations from different samples?

I greatly appreciated and looking forward to hearing back from you.

Sincerely,


Hyong Woo Choi

Post-Doc
Boyce Thompson Institute
533 Tower Road
Ithaca, NY 14850
e-mail:hc746@cornell.edu

You are right, sorry for the inconvenience caused due to the mistake. The buffer was prepared 1.7M sucrose diluting 1/2 from a 3.14M sucrose stock prepared by heating meanwhile stirring.

I found that the sucrose concentration in Buffer C was actually 1.7 M, but not 3.7 M, from the original plant journal article. Please correct this typo error.