PBMC samples chromatin extraction - low concentration

Rosmano · 01-07-2022, 06:06 AM

Hello,

I have been trying to extract chromatin from an immune system population, but the DNA concentration levels that I've been getting are extremely underwhelming.

Namely, from 1 million to 3 million cells I am getting concentrations of around 0.525ng/uL to 2.08ng/uL of chromatin in 50uL of SDS lysis buffer (1%SDS, 100mM EDTA, 50mM Tris pH 8.0), measured on Qubit with the dsDNA HS kit.
Fixation was done with Formaldehyde 37% for 10 minutes.

Anyone with experience on extracting chromatin from immune cells? I need to know if these are normal concentration values for these cells, because I am at a loss already.

Rosmano · 01-13-2022, 07:15 AM

Update: Tested a new formaldehyde to compare with the old one, but still got around the same concentrations. Tested this in total thymocytes instead of doing it on sorted cells. Still no improvement.

Update 2: Sonicated these samples. After sonication the concentration increased to the expected values. Uh. Apparently the sonication is required not only to fragment DDNA but to actually release from the cell debris?

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01-07-2022, 06:06 AM	#1
Rosmano Member Location: Portugal Join Date: May 2012 Posts: 23	PBMC samples chromatin extraction - low concentration Hello, I have been trying to extract chromatin from an immune system population, but the DNA concentration levels that I've been getting are extremely underwhelming. Namely, from 1 million to 3 million cells I am getting concentrations of around 0.525ng/uL to 2.08ng/uL of chromatin in 50uL of SDS lysis buffer (1%SDS, 100mM EDTA, 50mM Tris pH 8.0), measured on Qubit with the dsDNA HS kit. Fixation was done with Formaldehyde 37% for 10 minutes. Anyone with experience on extracting chromatin from immune cells? I need to know if these are normal concentration values for these cells, because I am at a loss already.

01-13-2022, 07:15 AM	#2
Rosmano Member Location: Portugal Join Date: May 2012 Posts: 23	Update: Tested a new formaldehyde to compare with the old one, but still got around the same concentrations. Tested this in total thymocytes instead of doing it on sorted cells. Still no improvement. Update 2: Sonicated these samples. After sonication the concentration increased to the expected values. Uh. Apparently the sonication is required not only to fragment DDNA but to actually release from the cell debris? Last edited by Rosmano; 01-13-2022 at 09:40 AM.