chromatin yield (ChIP) - chromatin yield from primary cells (Jul/04/2007 )
Hi all!
I have an urgent question for the ChIP experts among you 
I do ChIP experiments for some months now, but recently I faced a problem with the chromatin yield when I lysed and sonicated very small primary cells.
Does anyone of you know how much chromatin I 'should' get from, let's say 10 million cells that I fixed with formaldehyde, lysed and sonicated and did the reversal of the cross-link????
Has anyone of you experience with primary cells that are very small?
I actually don't know, if the poor chromatin yield is due to an incomplete lysis or if I have different problems (and what problems that might be?!). If I check the lysed cells after trypanblue staining by microscope, they are all stained blue. So I would say, that the lysis is ok (I lyse quite long, for 30 min on ice).
Many thanks in advance!
-positive thinking-
QUOTE (positive thinking @ Jul 4 2007, 08:51 AM)
Hi all!
I have an urgent question for the ChIP experts among you
I do ChIP experiments for some months now, but recently I faced a problem with the chromatin yield when I lysed and sonicated very small primary cells.
Does anyone of you know how much chromatin I 'should' get from, let's say 10 million cells that I fixed with formaldehyde, lysed and sonicated and did the reversal of the cross-link????
Has anyone of you experience with primary cells that are very small?
I actually don't know, if the poor chromatin yield is due to an incomplete lysis or if I have different problems (and what problems that might be?!). If I check the lysed cells after trypanblue staining by microscope, they are all stained blue. So I would say, that the lysis is ok (I lyse quite long, for 30 min on ice).
Many thanks in advance!
I have an urgent question for the ChIP experts among you
I do ChIP experiments for some months now, but recently I faced a problem with the chromatin yield when I lysed and sonicated very small primary cells.
Does anyone of you know how much chromatin I 'should' get from, let's say 10 million cells that I fixed with formaldehyde, lysed and sonicated and did the reversal of the cross-link????
Has anyone of you experience with primary cells that are very small?
I actually don't know, if the poor chromatin yield is due to an incomplete lysis or if I have different problems (and what problems that might be?!). If I check the lysed cells after trypanblue staining by microscope, they are all stained blue. So I would say, that the lysis is ok (I lyse quite long, for 30 min on ice).
Many thanks in advance!
I don't think the cell size should have any effect on the amount of chromatin you get. Any difference in size between nuclei of different cells is going to be a difference in the amount of soluble proteins and aqueous space, not in the actual chromatin (assuming we're NOT talking about droso salivary gland cells with polytene chromosomes or something similar).
Regarding your question of the chromatin yield from 10E06 cells, what are you using to measure the amount of chromatin? Absorbance at 260? Running PCR on the extracted DNA?
-KPDE-
QUOTE (KPDE @ Jul 5 2007, 12:01 PM)
QUOTE (positive thinking @ Jul 4 2007, 08:51 AM)
Hi all!
I have an urgent question for the ChIP experts among you
I do ChIP experiments for some months now, but recently I faced a problem with the chromatin yield when I lysed and sonicated very small primary cells.
Does anyone of you know how much chromatin I 'should' get from, let's say 10 million cells that I fixed with formaldehyde, lysed and sonicated and did the reversal of the cross-link????
Has anyone of you experience with primary cells that are very small?
I actually don't know, if the poor chromatin yield is due to an incomplete lysis or if I have different problems (and what problems that might be?!). If I check the lysed cells after trypanblue staining by microscope, they are all stained blue. So I would say, that the lysis is ok (I lyse quite long, for 30 min on ice).
Many thanks in advance!
I have an urgent question for the ChIP experts among you
I do ChIP experiments for some months now, but recently I faced a problem with the chromatin yield when I lysed and sonicated very small primary cells.
Does anyone of you know how much chromatin I 'should' get from, let's say 10 million cells that I fixed with formaldehyde, lysed and sonicated and did the reversal of the cross-link????
Has anyone of you experience with primary cells that are very small?
I actually don't know, if the poor chromatin yield is due to an incomplete lysis or if I have different problems (and what problems that might be?!). If I check the lysed cells after trypanblue staining by microscope, they are all stained blue. So I would say, that the lysis is ok (I lyse quite long, for 30 min on ice).
Many thanks in advance!
I don't think the cell size should have any effect on the amount of chromatin you get. Any difference in size between nuclei of different cells is going to be a difference in the amount of soluble proteins and aqueous space, not in the actual chromatin (assuming we're NOT talking about droso salivary gland cells with polytene chromosomes or something similar).
Regarding your question of the chromatin yield from 10E06 cells, what are you using to measure the amount of chromatin? Absorbance at 260? Running PCR on the extracted DNA?
Hi KPDE!
And thanks for your fast reply!
After I did the removal of the cross-link, I purify the aliquot and meassure the concentration of the DNA with a NanoDrop (--> absorbance at 260).
Do you think, that the lysis might be 'too good', leading to lysed nuclei and therefore a loss of chromatin?
-positive thinking-
QUOTE (positive thinking @ Jul 7 2007, 01:48 AM)
QUOTE (KPDE @ Jul 5 2007, 12:01 PM)
QUOTE (positive thinking @ Jul 4 2007, 08:51 AM)
Hi all!
I have an urgent question for the ChIP experts among you
I do ChIP experiments for some months now, but recently I faced a problem with the chromatin yield when I lysed and sonicated very small primary cells.
Does anyone of you know how much chromatin I 'should' get from, let's say 10 million cells that I fixed with formaldehyde, lysed and sonicated and did the reversal of the cross-link????
Has anyone of you experience with primary cells that are very small?
I actually don't know, if the poor chromatin yield is due to an incomplete lysis or if I have different problems (and what problems that might be?!). If I check the lysed cells after trypanblue staining by microscope, they are all stained blue. So I would say, that the lysis is ok (I lyse quite long, for 30 min on ice).
Many thanks in advance!
I have an urgent question for the ChIP experts among you
I do ChIP experiments for some months now, but recently I faced a problem with the chromatin yield when I lysed and sonicated very small primary cells.
Does anyone of you know how much chromatin I 'should' get from, let's say 10 million cells that I fixed with formaldehyde, lysed and sonicated and did the reversal of the cross-link????
Has anyone of you experience with primary cells that are very small?
I actually don't know, if the poor chromatin yield is due to an incomplete lysis or if I have different problems (and what problems that might be?!). If I check the lysed cells after trypanblue staining by microscope, they are all stained blue. So I would say, that the lysis is ok (I lyse quite long, for 30 min on ice).
Many thanks in advance!
I don't think the cell size should have any effect on the amount of chromatin you get. Any difference in size between nuclei of different cells is going to be a difference in the amount of soluble proteins and aqueous space, not in the actual chromatin (assuming we're NOT talking about droso salivary gland cells with polytene chromosomes or something similar).
Regarding your question of the chromatin yield from 10E06 cells, what are you using to measure the amount of chromatin? Absorbance at 260? Running PCR on the extracted DNA?
Hi KPDE!
And thanks for your fast reply!
After I did the removal of the cross-link, I purify the aliquot and meassure the concentration of the DNA with a NanoDrop (--> absorbance at 260).
Do you think, that the lysis might be 'too good', leading to lysed nuclei and therefore a loss of chromatin?
If you crosslinked then I don't think it would be very easy to "lyse" the nuclei. During lysis you should get rid of the nuclear envelope but the lamina should be intact (because of the crosslinking) and therefore, so should the chromatin. How are you determining that your yield isn't good? How many cells are you starting with, how much DNA do you end up with, and what are the dilution factors in between?
-KPDE-