ChIP assay - Foaming in sonication and other woes - (Jan/27/2005 )
Thanks for the quick reply. Im going to try a 10% setting tomorrow. I ran 5ul from my dna precipitation, starting with 1 million THP-1 cells. I could see the DNA pellet in the epindorph tube so I only ran 5ul. Again, thanks for the quick reply.
Thanks for the tips guys. I did many conditions on the sonicator we have...I can post the picture later if anyone likes. I found that increasing the volume to 400ul from 200ul helped the most with the resutls. 200ul isnt enought, it ends up foaming. Foaming = no chromatin shearing.
Dear all, I am working with a model of multiple sclerosis, I induce the disease in my mices with an emulsion. I use a water bath sonicator (bransonic) to emulsify 1:1 complete freud's adjuvant and MOG peptide in PBS, but the last time I could not get a stable emulsion, normally 15 minutes of sonication are enought to get a god emulsion but now I really do not what is going on with it. Please if you have information or an idea about this problem please let me know!!!!
I've just started using sonication to shear lengths of DNA, ideally between 200 and 1000bps, from transgenic murine T cells cultured in vitro for 10 days. When I run the products on an agarose gel to check the lengths, I get really beautiful laddering - sadly, I think this indicates that my cells are apoptosing. The very first step of the protocol (from Upstate) is to add, to a final concentration of 1%, formaldehyde to my cells. This should fix and kill the cells and I'm fairly sure that they're not undergoing apoptosis before I start the protocol. I don't get this pattern from normal murine ex vivo spleen cells. Any suggestions?
Hi, so the T-cells are suspension cultured right?? Do they form blasts like jurkat cells do?? If so then maybe the problem is that some cells aren't seeing formaldehyde quickly enough????
i usually see a smear, not laddering from sonication, but I never used this cell type....
Try to seperate the cells more and I would suggest that you keep the suspension of cells in say 5mL of CM, then take another 5mL of CM and add 2% FA then mix together to prevent localized high concentrations...
I hope that helps you...
Thanks for your suggestions. Yes, they do blast, but they are adherant cells. They are quite easy to remove from the wells so I have tried to remove excess medium (hoping to remove the majority of dead cells) then pipetted 1ml of medium per well (of a 24 well plate, 10^6 cells per well) up and down to remove the cells from the wells and into 1.5ml Eppendorfs. I then add the formaldehyde to the Eppies (final concentration 1%), and, as per the Upstate protocol, I incubate at 37 degrees for 10 minutes then wash the cells twice with PBS (and protease inhibitors). This protocol works really well for the fresh spleen cells and some cultured macrophages that I tried last week. I will try your suggestion of adding the formaldehyde to improve mixing, this seems like a likely solution and it could just be that my T cells are more susceptible to apoptosis than the other types that I have tried.
hi everybody, I just started to do the CHIP assay, I also have problems about shearing the chromatin DNA. so I join this forum for help
the following is my procedure.
1: I isolated the hippocampus of rat then minced in a 35mm plate filled 4 ml cold PBS
2: then I add the Formadehyde to a final concentraton of 1%
3: I incubate the plate at 37 C for 15 minutes(because the PBS is cold, so prolonged the incubation time)
4: I isoated the nuclear then lysis it in SDS lysis buffer.
5: and then I sonicate the lysis, the total volume I used is 800 ul.
I tried many sonicte conditions, the power setting range from 20% to 30% (KS 600 sonictor) the total time range from 60sec to 180sec (usually I will sonicate 20sec or 15sec, and then brake for 2min), I must note that there is no foaming when I did it.
but the sheared DNA is not good. there is a bright band at 4KD.
I am very frustrated about it
in addition, I found that the bad shearing will not enfluence the CHIP relults, I mean I can also get a positive band after PCR about 30 cycles (but not in the no antibody control), why this happened?
hope for your help
Is a water bath sonicator good for shearing DNA in a CHIP assay??. I use "SUPER RK 103H from Schütt labortechnik, i sonicate DNA at 100 strenght for 2 mins + 2 mins in ice cold water (i place the samples on ice after the first 2 mins). The DNA smear on the gel looks the same with sonication strenghts of 1.5+1.5 mins or 2+2 mins or 3+3mins. Could you please explain.
I am having similar trouble with ChiP sonication step. I follow the protocol from Santa Cruz, and use the reagents as per manufacture protocol. However, when I sonicated my 293T cell nuclear extracts reversed cross-link and phenol extracted the DNA, I only got a smear of DNA.
I used the following conditions:
cross-linking: 1% formaldehyde - 10min, 37oC
quenching: 0.125M Glycine.
mosonix 3000 with microtip, 15sec, 12X, at 1.5 continous output setting.
reverse: 65oC water bath 4hrs.
phenol extract, then RNase Treat 37oC 30min (did not perform protein K digest as phenol removes most of the protein.)
I am not sure what I am doing wrong. any suggestions?
A smear of DNA is exactly what you should expect.
The point is the smear should be in the range of 200 - 1000 bp.