ChIP sonication problem (View forum version)



krystle

Posted 16 February 2011 - 12:29 AM

Hi guys, i've been trying to optimise the sonication conditions for my cell type. When i tried to run gel to check the smear size, i get wierd looking results and i don't know what to make do of it. Will appreciate if someone can look at my gel and give me some advice. I've uploaded 2 picture of the same gel, one of which is overexposed. The first 2 lanes are ladders (1kb ladder, 100bp ladder), and followed by 6 lanes of samples (5 rds, 10rds, 15rds, 20rds, 25rds, 30rds)

What i did was to sonicate 200ul of samples (containing 2million cells). After 5rounds of 30s on/30s off (biorupter), i'll aliquot 20ul out. The remaining samples i'll continue to sonicate, and another 5 rounds later i aliquot 20ul again as the 10rd sample and so on. I repeat this until finally i have 100ul left which will be sonicated for another 5 rounds to make a total of 30 rounds.

After that i added RNase A and reverse the cross links, purify the pcr product using purification kit, and load the sample onto gel. The protocol i used was adapted from the abcam protcol, and apparently has been successful with my colleague on another cell type. I've quoted the protcol here,

"The DNA is purified using a PCR purification kit (add 70 μl of Elution Buffer and proceed to Step 3.2a)

3.2.a. Add 2 μl RNase A (0.5 mg/ml). Heat with shaking at 65°C for 4-5 hr (or overnight) to reverse the cross-links. DNA is purified using a PCR purification kit according to the manufacturer’s instructions. The samples can be frozen and stored at -20°C.

Samples are treated with RNase A as high levels of RNA will interfere with DNA purification when using the PCR purification kit. Yields can be severely reduced as the columns become saturated."


And just to clarify, the 30rds sample were eluted in the same amount even though there were 100ul of sample. So since i loaded equal volume for all, there were more 30rds samples and hence it looks much brighter. But still i can't explain why it looks like there is a band in the 200bp region.
And also, why don't i see the change in smear size as the no. of rounds of sonication increase?
Please help!!

  • 2011-02-16 CHIP.JPG
  • 2011-02-16 CHIP OVEREXPOSED.JPG

chabraha

Posted 16 February 2011 - 09:24 AM

krystle,

Your shearing looks fine to me. I would say 5-10 rounds is your target region. I think that the reason that you don't see the shearing go down until 30 rounds is cell-type specific according to your power setting. For example, in my case if I use a power setting of 2 on my sonicator I can shear all day but the DNA fragments will never become smaller than around 2kb. But, If I set my power to 8 I can have the DNA sheared to mononucleosomes ~200bp within 10 rounds. I think that your last band is a mononucleosomal band and that by decreasing your volume by 1/2 making your sonication much more efficient.

krystle

Posted 17 February 2011 - 12:09 AM

krystle,

Your shearing looks fine to me. I would say 5-10 rounds is your target region. I think that the reason that you don't see the shearing go down until 30 rounds is cell-type specific according to your power setting. For example, in my case if I use a power setting of 2 on my sonicator I can shear all day but the DNA fragments will never become smaller than around 2kb. But, If I set my power to 8 I can have the DNA sheared to mononucleosomes ~200bp within 10 rounds. I think that your last band is a mononucleosomal band and that by decreasing your volume by 1/2 making your sonication much more efficient.


Hi, thanks for your comments. After seeing what you posted, i decided to repeat the sonication just to confirm. This time round i started with 200ul sample, and aliquoted out 30ul after 10rds and 15rds. The remaining samples were subjected to 20 rds, after which 60ul was aliquoted out for the subsequent procedure

The loading for the gel is as follows, 1kb ladder, 100bp ladder, 10rds, 15rds, 20rds.

This time round, i see a bright band around 500bp region. Since i keep seeing the bright bands at the last samples i prepared where i always loaded more, i'm beginning to wonder if the band has anything to do with me overloading the column or the reaction or something.

Anyways, based on this gel, the 15 rds one still seem to be a bit on the bigger side (400bp -1.4kb)? So i'm thinking i probably need to try again at 20 or 25 rounds? Or is the 15rounds size acceptable already?

Thanks for your help!

  • 2011-02-17 chip less zoom.JPG

chabraha

Posted 17 February 2011 - 08:47 AM

I would agree, I didn't realize you were loading that much material onto the gel. As for your shearing, it's definitely a matter of opinion, and in my opinion I would say 15 rounds is enough. Like I said though it's what you feel comfortable with. I think that the least amount of shearing you can do but still get your controls to work is the best option since more shearing exposes the protein/DNA complexes to more stress. I also believe that how you analyze your data allows for larger fragments, as your negative control region will increase but your specific region of interest will increase as well, so as long as you account for this in your calculations (ie- divide specific IP/negative region IP) your data will be good to go.