ChIP - Foaming in sonication and other woes
Posted 27 January 2005 - 11:12 AM
Also, which type of vessel do you use for sonication? I use 2 ml eppendorf tubes, but the instruction manual of my sonicator recomends metal or glass vessels, better than plastic ones, because of heating considerations.
And last but not least, have you noticed differences in the sonication quality when using different cell lines? For some reason, I get really small fragments with 3T3 cells but not with 10T/2, and both of them are fibroblast cell lines. I don't change the conditions and sometimes I perform the experiments in parallel
Any thoughts or suggestions will be very welcome. Thanx!
Posted 27 January 2005 - 11:57 AM
To avoid foaming, dip the probe all the way to the bottom of the tube.
To avoid heating, hold the tube in ice water during sonication.
Hope that is useful.
Edited by pcrman, 27 January 2005 - 11:59 AM.
Posted 28 January 2005 - 07:19 AM
Posted 15 February 2005 - 12:18 PM
Contact Misonix and they will be able to provide lab procedures for DNA shearing using the cup horn.
Posted 16 February 2005 - 02:23 PM
After 10 sec on the first sonication pulse a get lots of foaming, but I like to do longer pulses (20 sec) and foaming is supposed to denature the sample. For how long do you sonicate, and how many pulses? I'm using a Branson 250 sonicator.
We use a Branson Sonifier B-30. I had real problems with foaming initially and I came to realise that the chromatin is not shearing properly when it foams up. We were using maximum settings on the sonifier with no luck.
So I tried lowering the settings with the same pulse times. The settings I used and were sucessful are a duty cycle of 30% and power setting to 3. I performed 15 continuous pulses for ten seconds each in 1.5 eppy tubes set in an ice bath to reduce the heat from sonication. We resuspend and sonicate our chromatin in RIPA buffer also.
I would suggest lowering the settings of the sonicator, that certainly stopped the foaming issue and still sheared the chromatin quite nicely.
The cells we work with are CHO hybrids but I am sure different cell lines behave differently. You could try optimising formaldehyde fixation times as I would think cell lines would behave differently to the fixation. (for our cells, 1% formaldehyde in media for 10 minutes exactly is suffice)
hope this helps!
Posted 18 February 2005 - 07:58 AM
Nick, I recently tried something similar to what you said: I fixed the cells using a lower concentration of formaldehyde than usual (0.5% for 10 min) and the shearing seemed to improve. I'll try your suggestion to decrease the power of the sonicator and increase the number of pulses.
Sonix, I thought water bath sonicators are not fit for shearing the DNA for ChIP assays, 'coz they're not powerful enough. I checked the webpage you indicated and it says that this cup horns are high intensity sonicators, but will the sample not heat up a lot during sonication? Heating of the sample during sonication, I heard, is supposed to denature the proteins, even break the formaldehyde bonds.
Thanx again both of you for your help!
Posted 16 March 2005 - 11:54 PM
Nick: I would suggest lowering the settings of the sonicator, that certainly stopped the foaming issue and still sheared the chromatin quite nicely.
Woe, this tip works and solved my foaming problem too! Previously I used a power setting of 15% (setting 3) and got foaming easily. After I lowered the setting to 10% (setting 2), I hardly got any foaming. Thank you Nick!
Here is my result with high and low power (volume 400 ul)
Edited by pcrman, 23 March 2005 - 12:55 PM.
Posted 17 March 2005 - 01:55 PM
I always thought that you needed the high setting to ensure proper chromatin shearing. but you don't
Posted 23 March 2005 - 11:19 AM
I used a 25ml sample in a sonication test on duty-60%, intensity=6 for 5 minutes, taking samples every minute. Running the crude extract and the insoluble suspension on an SDS-PAGE gel for each time point showed very little difference in sonicating one minute and sonicating five minutes.
In addition, the insoluble and soluble bands were identical, which makes the presence of inclusion bodies less likely.
My lab is relatively new to sonication, so the hope is that the error is ours and not in the experiment. If sonication produced foaming, therefore denaturing the proteins, would this explain the results - at least in part?
Posted 23 March 2005 - 12:14 PM
Here we are talking about sonicating DNA. It seems that you are sonicating protein, right?
Once foaming occurs, sonicating efficiency goes almost to zero. I found your power setting is very high, have you experienced foaming? If yes, decrease your setting. Your are using 25 ml sample for sonication, that sounds to much to me. For ChIP sonication, the max volume I tried is 400 ul. Certainly the bigger the volume, the lower the efficiency.
Posted 23 March 2005 - 02:35 PM
there have been some changes to the website, and the inclusion of photos with the messages is just awesome!!!
great to see lowering the settings worked for ya! seems so simple a change doesn't it?
Posted 28 March 2005 - 05:09 PM
1) When taking the agarose beads using a yellow tip, cut off its end otherwise you will end up with dry beads only left (because the diameter of the beads is roughly the same of lumen of tip end.
2) After reverse crosslinking at 65C for 4 hr and one hour of proteinase K digestion at 45C, before going to phenol/chloroform extraction, you have to transfer the samples into a new tube because the cap no longer fit tightly on the tube after intensive heating. If you don't, your sample will leak when you vortex.
Posted 29 March 2005 - 12:59 AM
There are many nice man tell me the tricky point in experiment...
These help me to improve my exp.
Thanks very much, Everybody...