8 replies to this topic

[Mighty Mouse]

I had a quick question. I'm still a relative newbie at ChIP and want to ensure that I'm not encountering anything odd before moving forward with my experiments.

Following sonication (I use a BioRuptor for 5 min) and after freezing samples at -80 and thawing on ice, there is a white precipitate at the bottom of my eppy tube. Is this to be expected? If so, what is it? And should I be spinning my samples down prior to removing chromatin for the IP step?

Thanks.

MM

[Clare]

Hello

Does your nuclear lysis buffer contain 1% SDS? If so, that white stuff could be SDS. After sonication I spin the samples at max speed for 10 mins (I set the centrifuge to 8deg so it does not get too cold and precipitate out the SDS)  then use the SN in ChIP

Clare

Mighty Mouse, on Dec 9 2009, 02:57 AM, said:

I had a quick question. I'm still a relative newbie at ChIP and want to ensure that I'm not encountering anything odd before moving forward with my experiments.

Following sonication (I use a BioRuptor for 5 min) and after freezing samples at -80 and thawing on ice, there is a white precipitate at the bottom of my eppy tube. Is this to be expected? If so, what is it? And should I be spinning my samples down prior to removing chromatin for the IP step?

Thanks.

MM

[gogreen]

I used to see this during my ChIP days too..I guess its just the gravity settling cell components upon storage. not to worry, quite natural!

You have to centrifuge the sample before taking the chromatin for IP. I use the centrifuge at 4 degrees or carry out the experiment in a cold room. Its better to work with the tubes on ice as much as possible.

all the best!

[Mighty Mouse]

Thanks for your responses. I feel better about my samples now! I'll spin them down in the future and take off the supernatant.

[Clare]

YAY!

Mighty Mouse, on Dec 9 2009, 03:11 PM, said:

Thanks for your responses. I feel better about my samples now! I'll spin them down in the future and take off the supernatant.

[Mighty Mouse]

Just out of curiosity, do you know if having a high concentration of SDS during immunoprecipitation can cause high background? I'm currently have an issue with very high background in my samples (e.g, my IgG controls), and I wonder if it's because I didn't spin them down prior to removing the chromatin for the IP.

[Clare]

You shouldn't have a high SDS conc. during the actual IPs - don't you dilute your sample before adding antibodies etc?

How many cells are you using per IP and how much antibody/IgG do you add?

And what exactly is 'very high background'? Ct?

Clare

Mighty Mouse, on Dec 11 2009, 12:50 AM, said:

Just out of curiosity, do you know if having a high concentration of SDS during immunoprecipitation can cause high background? I'm currently have an issue with very high background in my samples (e.g, my IgG controls), and I wonder if it's because I didn't spin them down prior to removing the chromatin for the IP.

[Mighty Mouse]

[quote name='Clare' date='Dec 15 2009, 12:11 PM' post='51460']
You shouldn't have a high SDS conc. during the actual IPs - don't you dilute your sample before adding antibodies etc?

How many cells are you using per IP and how much antibody/IgG do you add?

And what exactly is 'very high background'? Ct?

Clare

Clare,
  I do dilute my samples for the IP (2 ug of DNA into 500 uL), but I guess my SDS concentration was way too high. My background before when I didn't spin down my samples hit Ct's around the mid to high 20's! I spun down my samples and re-ran everything and the mock samples were in the 33-36 Ct range, which seems to be much more in line with what people get and what I've gotten before. I add about 1-2 ug of IgG and 0.1 to 2 ug of antibody (depending on manufacturer's recommendation etc...).

As for the cells, I actually use about 30 mg of brain tissue (the hippocampus); not quite sure what the number of cells actually is, but it's enough to get a working ChIP (sometimes at least!).

Anyway, it seems the lack of centrifugation prior to taking some of my sample for the IP was the problem as my background has dropped down to more "normal" levels.

MM

[Clare]

That's good to hear

Clare

Clare,
  I do dilute my samples for the IP (2 ug of DNA into 500 uL), but I guess my SDS concentration was way too high. My background before when I didn't spin down my samples hit Ct's around the mid to high 20's! I spun down my samples and re-ran everything and the mock samples were in the 33-36 Ct range, which seems to be much more in line with what people get and what I've gotten before. I add about 1-2 ug of IgG and 0.1 to 2 ug of antibody (depending on manufacturer's recommendation etc...).

As for the cells, I actually use about 30 mg of brain tissue (the hippocampus); not quite sure what the number of cells actually is, but it's enough to get a working ChIP (sometimes at least!).

Anyway, it seems the lack of centrifugation prior to taking some of my sample for the IP was the problem as my background has dropped down to more "normal" levels.

MM
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