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SDS, Cell numbers, dilution


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#1 MunkySpunk

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Posted 28 July 2009 - 06:02 AM

Hi guys, I've been reading this board and it's a wealth of information on this method. Thank you all for your help both in the past and in advance.

Like most people, I'm using the upstate kit to perform my first ChIPs, and like most people I'm running into problems. Don't even get me started on Upstate's fast-talking about how they give you enough reagents for 22 IP's but only enough for 10 lysis/sonication events.

I'm using LNCaP cells and searching for nuances in p53 acetylation at the p21 locus upon HDAC inhibitor treatment. LNCaP cells are very very picky, and they do not like to be too crowded (or too sparse for that matter) in the culture dish. I can fit 10e6 cells in a 10cm dish, possibly 2X10e6 if I'm pushing it. Scaling up, I can get 4.5X10e6 cells into a 15cm dish, but that's REALLY pushing the limits of these cells. If I'm talking 96 hour timepoints, I can't put any more than 10e6 into the plate or they overgrow and start dying - as you can imagine, this kind of screws with the p53 status of the cell.

But this relates to my problem: If I'm using 10e6 cells/dish/sonication, I don't have any capacity to dilute the SDS lysis buffers in ChIP dilution buffer. This means I'm heading into the IP steps at a full SDS concentration. Won't this interfere with the antibody binding?

The protocol they supply is built around 10e7 HeLa cells and they only give passing remarks on what to do in other cases, with none of those remarks actually providing any solutions.

Has anybody successfully and reliably performed ChIP on a sample without usng the ChIP dilution buffer? Does anyone have any suggestions here?

Edited by MunkySpunk, 28 July 2009 - 06:02 AM.


#2 KPDE

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Posted 28 July 2009 - 08:05 AM

Hi guys, I've been reading this board and it's a wealth of information on this method. Thank you all for your help both in the past and in advance.

Like most people, I'm using the upstate kit to perform my first ChIPs, and like most people I'm running into problems. Don't even get me started on Upstate's fast-talking about how they give you enough reagents for 22 IP's but only enough for 10 lysis/sonication events.

I'm using LNCaP cells and searching for nuances in p53 acetylation at the p21 locus upon HDAC inhibitor treatment. LNCaP cells are very very picky, and they do not like to be too crowded (or too sparse for that matter) in the culture dish. I can fit 10e6 cells in a 10cm dish, possibly 2X10e6 if I'm pushing it. Scaling up, I can get 4.5X10e6 cells into a 15cm dish, but that's REALLY pushing the limits of these cells. If I'm talking 96 hour timepoints, I can't put any more than 10e6 into the plate or they overgrow and start dying - as you can imagine, this kind of screws with the p53 status of the cell.

But this relates to my problem: If I'm using 10e6 cells/dish/sonication, I don't have any capacity to dilute the SDS lysis buffers in ChIP dilution buffer. This means I'm heading into the IP steps at a full SDS concentration. Won't this interfere with the antibody binding?

The protocol they supply is built around 10e7 HeLa cells and they only give passing remarks on what to do in other cases, with none of those remarks actually providing any solutions.

Has anybody successfully and reliably performed ChIP on a sample without usng the ChIP dilution buffer? Does anyone have any suggestions here?


You could always try a sonication buffer that doesn't have SDS. The ChIP method is pretty robust and will handle changes in buffers. The buffer we use is: 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.5% (vol/vol) NP-40 (a.k.a. IGEPAL), 1.0% (vol/vol) Triton X-100, plus protease and phosphatase inhibitors. Also, don't be afraid to use fewer cells. If you are using magnetic beads you could probably go down to 5 X10^5 and if using magnetic beads with the Fast ChIP method, probably down to 1 X 10^5 (this is what I have used).

#3 MunkySpunk

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Posted 28 July 2009 - 10:51 AM

What volume do you generally sonicate in and what's the density of the cells in said volume?

I'm using 5X10e5 in 500ul and I'm getting good sonication after 8X10s pulses. I'll obviously have to do a shearing assay to optimize the fragment size for the new buffer, but can you give me a ballpark figure? Does your lysis buffer tend to require more or less sonication than its SDS counterpart?

#4 KPDE

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Posted 28 July 2009 - 02:30 PM

What volume do you generally sonicate in and what's the density of the cells in said volume?

I'm using 5X10e5 in 500ul and I'm getting good sonication after 8X10s pulses. I'll obviously have to do a shearing assay to optimize the fragment size for the new buffer, but can you give me a ballpark figure? Does your lysis buffer tend to require more or less sonication than its SDS counterpart?


I was doing about 5-10 X10e6 cells per ml. I never compared sonication in the buffer I use to buffer with SDS but I have heard that SDS can increase sonication efficiency. So it's possible you would need to increase your sonication time.

#5 epi123

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Posted 28 July 2009 - 07:14 PM

SDS definitely affects ChIP efficiency. When a ChIP is working, the SDS final concentration is not greater than ~0.3%.
i'm using a kit which recommends sonicating 1million cells in 50ul of lysis buffer. then dilute this to the number of cells you want to use per ChIP. On averge 150,000 cells per ChIP works really well.

hope this helps

#6 MunkySpunk

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Posted 31 July 2009 - 05:03 AM

If that's the case (and it sounds quite plausible), the SDS concentrations are probably leading to my ChIP inconsistencies.

The Triton and the Nonidet P-40 in the alternative lysis buffer won't interfere with antibody binding, will they?

As far as the Chelex method for eluting DNA: Does it give a better yield than Phenol/Ethanol/Carrier-RNA for elution? It's quite obviously faster and easier, but in my experience, that usually doesn't equate to better.

Thanks for all your help.

EDIT: Looking at the Fast ChIP protocol, two lysis steps are required - one for cell, followed by spin and aspirate, then another with the same buffer, for nuclear lysis I would assume.

As both these steps rely on the same buffer, does anyone have any strategies to minimize nuclear lysis with the first step so as to ensure I recover as much genomic DNA as possible (as opposed to aspirating it up after the first spin)? Is it a function of time? i.e. do I just pipette a few times and throw it directly into the 'fuge?

Edited by MunkySpunk, 31 July 2009 - 05:42 AM.


#7 epi123

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Posted 31 July 2009 - 06:03 AM

as for chelex...it's faster than phenol; however sometimes it's hard to keep the same amount cosistent from sample to sample. invitrogen's chip kit it uses magnetic beads for DNA recovery which helps with yield.

#8 KPDE

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Posted 31 July 2009 - 09:12 AM

as for chelex...it's faster than phenol; however sometimes it's hard to keep the same amount cosistent from sample to sample. invitrogen's chip kit it uses magnetic beads for DNA recovery which helps with yield.


A number of colleagues have begun using Fast ChIP with magnetic beads (e.g. Dynabeads). They get the speed and the low background. I haven't tried it yet, as I am mostly doing 96-well based ChIP, but it seems to be working very well for them.

#9 MunkySpunk

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Posted 31 July 2009 - 09:56 AM

Can I assume you're referring to Flanagin et al.?

I'm pretty keen to move onto the 96-well plate format ChIP once I get my own stuff working reliably, and maybe rig up a plate-sized electromagnet out of an old steel license plate or something for the washes. Out of curiosity, how many cells do you plate in each well for this format, and how many PCR reactions worth of DNA does each well yield?

I just got done using the above mentioned lysis buffer on a new set of cells and doing a battery of sonications to re-optimize. I'll post on the results (Sonicator settings as a function of SDS or Triton buffers) once I run the gel.

#10 epi123

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Posted 31 July 2009 - 10:27 AM

the invitrogen ChIP kit not only as dynabeads for the immunoprecipiation step but also has different magnetic beads in the kit that are used for the DNA recovery of the ChIP DNA.

#11 KPDE

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Posted 01 August 2009 - 10:48 AM

Can I assume you're referring to Flanagin et al.?

I'm pretty keen to move onto the 96-well plate format ChIP once I get my own stuff working reliably, and maybe rig up a plate-sized electromagnet out of an old steel license plate or something for the washes. Out of curiosity, how many cells do you plate in each well for this format, and how many PCR reactions worth of DNA does each well yield?


One of the benefits of doing the plate based ChIP with the Flanagin et al. method is that you don't have to use beads. We just bind the protein A directly to the wells. I didn't think there would be enough surface area but Steve found that high-bind polystyrene plates from Corning work great. They do give more background than magnetic beads (similar to using agarose beads) but I'll take that background if it means less sample transfers (i.e. keeping it in the plate is much simpler and easier). Typically we use 50,000-100,000 cells per well though the method still works down to 10,000 cells. Using the method as it is in the paper we elute in 100Ál so that should give you an idea how many PCR reactions you can do.

Let me know if you're interested and I can give you a detailed protocol and some tips to get you started. It is a little more involved for getting started than Fast ChIP (since Fast ChIP is essentially the same method everyone uses just with a small tweak at the end) but once you get it working you quickly see the advantage. A few times I harvested cells in the morning, sonicated just before noon, ran 150 ChIP samples (2 plates) in the afternoon, and loaded up a PCR plate before going home, and was much more labor than doing 24 ChIP samples with Fast ChIP starting with already sonicated chromatin (this is assuming you have a repeat pipettor or multi-channel pipettor for all the washes and blocking step).

Also, I'm curious what your sonication profiles will look like. Definitely post them. Good luck.

Joel

#12 MunkySpunk

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Posted 03 August 2009 - 09:06 AM

Tried the new ChIP buffer. The results didn't look very good. I performed 4 lysis/sonication reactions. Each in a volume of 500ul with 10e7 cells/ml (I split up 2X10e7 cells into 4 equal parts for sonication). The only sonication that gave me anything whatsoever as far as DNA in concerned was the sample I pulsed 12X10s at 75% power. The 12X10s pulses, coincidentally, gave me a smear where I wanted one, but none of the other lanes showed ANY DNA, much less a smear at the wrong size.

The fact that none of the other lanes from that particular 500ul sonication/lysis reaction showed ANY DNA leads me to conclude my problem was downstream of the sonication step. But I also did not get DNA in my no sonication negative control, which would lead one to clnclude that it's the steps where I use the new lysis buffer.

Here's what I do post-sonication: I spin down the sample as per step 8 of Nelson et al. 2006. I take that supernatant, add 1.5ul of RNAse A, 37 for 30 minutes, add 1.5ul of Prot K, 62 for 2 hours. After the Prot K step, I had lots and lots of precipitate, so I spun that down, added loading dye, and loaded 1/2 the sample without disturbing the pellet.

Is it possible that my DNA is in the pellet from the precipitate post-sonication and I just disturbed that one pellet a bit when loading the gel?

Am I utilizing the new lysis buffer properly? I add it, pipette, promptly spin, remove supernatant, add more, spin again, and call it lysed (as per steps 4-6 of Nelson et al.

I'm about to run the rest of my samples on another gel, pellet and all, to test my wild guess.

Any answers prior to me running this ge would be greatly appreciated.

Edited by MunkySpunk, 03 August 2009 - 09:10 AM.


#13 KPDE

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Posted 03 August 2009 - 11:43 AM

Tried the new ChIP buffer. The results didn't look very good. I performed 4 lysis/sonication reactions. Each in a volume of 500ul with 10e7 cells/ml (I split up 2X10e7 cells into 4 equal parts for sonication). The only sonication that gave me anything whatsoever as far as DNA in concerned was the sample I pulsed 12X10s at 75% power. The 12X10s pulses, coincidentally, gave me a smear where I wanted one, but none of the other lanes showed ANY DNA, much less a smear at the wrong size.

The fact that none of the other lanes from that particular 500ul sonication/lysis reaction showed ANY DNA leads me to conclude my problem was downstream of the sonication step. But I also did not get DNA in my no sonication negative control, which would lead one to clnclude that it's the steps where I use the new lysis buffer.

Here's what I do post-sonication: I spin down the sample as per step 8 of Nelson et al. 2006. I take that supernatant, add 1.5ul of RNAse A, 37 for 30 minutes, add 1.5ul of Prot K, 62 for 2 hours. After the Prot K step, I had lots and lots of precipitate, so I spun that down, added loading dye, and loaded 1/2 the sample without disturbing the pellet.

Is it possible that my DNA is in the pellet from the precipitate post-sonication and I just disturbed that one pellet a bit when loading the gel?

Am I utilizing the new lysis buffer properly? I add it, pipette, promptly spin, remove supernatant, add more, spin again, and call it lysed (as per steps 4-6 of Nelson et al.

I'm about to run the rest of my samples on another gel, pellet and all, to test my wild guess.

Any answers prior to me running this ge would be greatly appreciated.


The precipitate is interesting. Probably that's where your material is. I would consider skipping the EtOH precipitation step altogether. Just add your chromatin to 100ul chelex (if the volume is too large just spin down the chelex, remove the supe, and add your chromatin to the pellet of chelex resin), add proteinase K, digest and boil and run the supe on the gel.

#14 MunkySpunk

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Posted 03 August 2009 - 12:19 PM

I don't do EtOH precipitation. This is just a shearing test and my results are quite mind-boggling.

The DNA is not in the pellet portion of the sample. I just ran it on a gel and got the same one smear in a single lane with no DNA (smear or otherwise) anywhere else.

Again, what do you guys do, exactly, to lyse the cells with the IP buffer? I'm losing my DNA somewhere between lysis and running a smear on a gel. How many times do you pipette the first time (step 4 Nelson et al.)? How vigorously do you pipette? How long does it stay in this buffer before you spin it down? How vigorously do you resuspend the pellet in the second wash (step 6)?

I've got a picture of my mystery gel below. Each lane indicated the number of pulses each sample received and the duration of each pulse.

(-) is negative control. Just lysis buffer, no sonication.

The group of samples on the left were washed together in the same tube and sonicated together in the same tube (I sonicate twice, remove 50ul, and so on...) Same for the group on the right.

As you can see, my 12X10s pulse lane has DNA fragments of decent size, albeit a bit small. Given that all of the 10s pulse samples came from the same tube, were lysed together, and sonicated together, my gel is telling me that either the cells spontaneously grew DNA somewhere between 10 and 12 pulses, or the DNA is being lost after sonication somewhere.

Can anyone suggest what may be causing this? Where could my DNA possibly be hiding out.

Thank you all.

Attached Thumbnails

  • Shear_test_5_New_Buffer1.jpg

Edited by MunkySpunk, 03 August 2009 - 12:20 PM.


#15 KPDE

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Posted 03 August 2009 - 01:35 PM

I don't do EtOH precipitation. This is just a shearing test and my results are quite mind-boggling.

The DNA is not in the pellet portion of the sample. I just ran it on a gel and got the same one smear in a single lane with no DNA (smear or otherwise) anywhere else.

Again, what do you guys do, exactly, to lyse the cells with the IP buffer? I'm losing my DNA somewhere between lysis and running a smear on a gel. How many times do you pipette the first time (step 4 Nelson et al.)? How vigorously do you pipette? How long does it stay in this buffer before you spin it down? How vigorously do you resuspend the pellet in the second wash (step 6)?

I've got a picture of my mystery gel below. Each lane indicated the number of pulses each sample received and the duration of each pulse.

(-) is negative control. Just lysis buffer, no sonication.

The group of samples on the left were washed together in the same tube and sonicated together in the same tube (I sonicate twice, remove 50ul, and so on...) Same for the group on the right.

As you can see, my 12X10s pulse lane has DNA fragments of decent size, albeit a bit small. Given that all of the 10s pulse samples came from the same tube, were lysed together, and sonicated together, my gel is telling me that either the cells spontaneously grew DNA somewhere between 10 and 12 pulses, or the DNA is being lost after sonication somewhere.

Can anyone suggest what may be causing this? Where could my DNA possibly be hiding out.

Thank you all.


As far as lysis goes, I just pipet up and down enough to break up the pellet. I'm not to vigorous about it.

So, after sonication and clearing by centrifugation, what percentage of the original cell pellet would you say the resulting post sonication pellet is? For me it is typically 5% or less.




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