Hi Oscar,

So, when you start reverse crosslinking, you begin with adding 5M NaCl to a final conc of 0.2M to the elute. Keep this at 65C Overnight/Shaking. The pellet is due to some precipitated protein.

After overnight reverse cross-linking, add proteinase K to a final conc of 2mg/ml and incubate at 37C/2-3 Hours/Shaking. Add RNase A to a final conc of 2mg/ml and again incubate at 37C/2-3 Hours/Shaking. Finally, I would do Phenol Chloroform Extraction and then precipitate DNA. I avoid columns. This method has worked very well for me.

Hope this helps. Good Luck!

Utkarsh

If your problem is not getting enough DNA out of your IP'ed samples I'd start with extending your reverse crosslinking to an O/N incubation or even add some prot K. You're probably cutting it short with just 30 mins on 65 celsius and you probably aren'y getting all the DNA off of the beads you're using. In addition the protein still linked to your DNA might disturb the downstream column purification.  

If you're worried about the isolation kit then just perform an ethenol precipitation instead. 

Hope this helps

Hi Oscar,

So, when you start reverse crosslinking, you begin with adding 5M NaCl to a final conc of 0.2M to the elute. Keep this at 65C Overnight/Shaking. The pellet is due to some precipitated protein.

After overnight reverse cross-linking, add proteinase K to a final conc of 2mg/ml and incubate at 37C/2-3 Hours/Shaking. Add RNase A to a final conc of 2mg/ml and again incubate at 37C/2-3 Hours/Shaking. Finally, I would do Phenol Chloroform Extraction and then precipitate DNA. I avoid columns. This method has worked very well for me.

Hope this helps. Good Luck!

Utkarsh

Yes, you should do a proteinase K digest. Not sure about the gene jet columns, but with quiagen columns or (which ones I prefer) zymoresearch ChIP DNA clean & concentrator columns (cheap!) you don't have a problem.

My guess is that you have protein precipitation due to the high salt concentration. You cannot omit the NaCl since it helps decrosslinking proteins and DNA.

Add input sample: Keep it frozen and process it along with the ChIP samples.

Presence of stubborn white pellet removes the possibility of a highly dissoluble salt. It's either protein or may even be (surprisingly) some thing out of the column itself. So better cross check the column by putting only the buffer and binding buffer not xture in it and pass through.

If not column, it might be protein, try performing chloroform- iso amyl alcohol after reverse-crosslinking at a suitable step.

All the best!

Hi Oskar,

1) I used the EZ-ChIP kit from Millipore. The process is almost same, as you mentioned. I always get a white turbidity with binding reagent. According to manufacturer, the precipitate may be observed, but this will not interfere with this procedure. The statement is absolutely correct, since I never faced any problem with the precipitation. So, I think it should not be a problem for you also.

2) You should add RNase and Proteinase K., although there is no correlation with turbidity and Rnase/proteinase. The precipitation will appear even after adding those.

3) Why you add elution buffer immediately? Before addition of antibody, take out 1% supernatant (v/v) and keep it in 4oC. Next day, add elution buffer to the Input tubes and samples tube simultaneously. I do the same without any problem.

For detail method, you can follow the EZ-ChIP™ Instruction Manual.

All the best,

Nirmalya

Hi Oscar, 

I guess the mess you are making is basically in reverse cross linking ( who gave you this protocol....!!!!?), anyway use ChiP elution buffer [100mm NaHCO3 (sod. bicarbonate) and 1%SDS solution in dH2O] incubate minimum 8 hours ( I recommend to do that O/N) at 650 C and then using either PCR elution or Gel extraction kit from Qiagen elute your DNA. Hope this will help.

Utkarsh, thank you for the advises and the pellet information. Now I worry that some of the DNA remains attached to these proteins and miss out, so protease K should help in that part.

Kevin, do you mean that with these kits there is no need to perform the digestions? About the INPUT, with only one day of waiting, will be better freezing or just keep the simple at 4ºC?

Mala, has plenty sense what you say, because in the few cases I don`t get a pellet with the samples after the incubation, once I add the binding buffer, the pellet appears!

Nirmalya, did your kit advised to use isopropanol with fragments smaller tan 500bp? Mine does and when I add, the turbidity disappears, but not the pellet. It would not be a problem, but I cannot resuspend it, only make it in to small pieces and transfer to the column. Do you do the same or you can resuspend it?

I think you're right with the INPUT issue, tan you.

The problem with phenol extraction is the inhibitory effects that it could have in the qPCR, plus the variability caused (between samples) in the extraction of the aqueous phase.

However today I will make a test with columns and phenol; and with and without digestion. Because I just did an electrophoresis with chromatin decrosslinked past night and I see nothing. I did not added salt to half of the samples and the pellet still appeared, so it have to be the proteins.

Weirdest thing is that I did the same with that very same sample before freezing and storing it and, extraction went fine. I still got the pellet and maybe lost part of my DNA, but I deffinitely saw DNA in the electrophoresis.

I noticed that elution buffer contains a lot of EDTA. EDTA is soluble at pH 8.0, and may form pellet if pH becomes acidic. In case of PCR purification kit from Fermentas (ThermoFisher ), binding buffer is acidic, and should be acidic when mixed with PCR reaction (otherwise NaOAc pH5.2 should be added).

Next point. If EDTA goes with DNA to the end and finally contaminate the PCR reaction this would cause a very low signal. Avoid using EDTA containing buffer. You can use ChiP eluton buffer suggested by Hasan Rajabi.

Next point. In the end of ChIP protocol you will obtain a very small quantity of DNA. So small that is not enough to form good pellet to be absorbed on membranes in DNA purification kits. To increase DNA recovery, use carriers like tRNA or glycogen.

Dear Oscar:

The white precipitate is most probably the resin leaching from the spin column and should not interfere with downstream applications like quantitative PCR or it could be protein precipitating out.

I agree with the other contributors that you must reverse cross link for a longer time and should include RNase A digestion followed by a Proteinase K digestion followed by clean up with a reputable kit ... I have always used Qiagen MinElute kit for my ChIP reactions .(Cat. # 28204 for $139.00; 50 spin columns).

Excerpt from Active Motif ChIP Protocol follows:

The complete pdf of this protocol is available online at

http://www.activemotif.com/documents/1574.pdf

Email: [email protected]

Chromatin Elution Section of the Protocol is as follows:

1. Re-suspend the magnetic beads carrying chromatin bound to antibody (washed with Buffers-1 and -2 sequentially) in 50uL of Elution Buffer AM2.

2. Incubate at 24 degrees C for 15 min with tilt mixing or on a rotator.

3. Spin tubes briefly to bring down any buffer stuck to the inside of the tube caps.

4. To the beads now suspended in 50uL of Elution buffer, add 50uL of Reverse Cross-Linking Buffer and mix well by pipeting. Separate the magnetic beads on a magnetic stand and transfer the 100uL of “Eluted chromatin” (it is still cross-linked) to a clean tube.

5. Now incubate the 100uL of “Eluted chromatin” at 95 degrees C for 15 minutes in a Thermo Cycler.

OR

If your “Eluted chromatin” contains a lot of protein, you may incubate at 65 degrees C overnight (8 hours at 65 degrees C should be sufficient). I recommend you do this for your samples. Now the chromatin cross links have been removed.

6. Add 2 uL RNase A and digest at 37 degrees C for 1 hour.

7. Add 2uL Proteinase K and incubate at 37 degrees C for 1 hour. Then add 2uL of Prot. K Stop Solution.

8. Clean up the eluted and cross link reversed chromatin DNA by using Active Motif chromatin IP DNA Purification Kit (Cat. # 58002) or (my choice) Qiagen MinElute kit (Cat. # 28204 for $139.00; 50 spin columns) .

9. Use 2uL of cleaned up, reverse cross linked eluted chromatin per qPCR reaction.

I hope this helps

Dear Oskar,

No, they don't recommend isopropanol treatment. I load the sup with the suspended particles in the column directly.

Hello guys thank you very much for your answers I really appreciate it.

Hasan thanks for the advise, as Dmitry said I tried to repeat the extraction with your buffer (no EDTA); I tried different combinations.

I used 3 µL of 6 different chromatin samples  with a concentration of 0.5µg/µL approx. I diluted them with 97µL of elution buffer (Hasan version). Moreover in order to conclude if the precipitated was caused by EDTA,  I took 6 tubes and add 100µL off Hasan’s buffer in 3 and mine (with EDTA) in the other 3; and I incubate under the same conditions as the chromatin ones without enzymes:

The conditions were: Adding NaCl 0.2M, incubate overnight at 65ºC (+RNase 0.01µg/µL), and then 3 hours at 37ºC (+Proteinase K 0.05µg/µL). 

• Chromatin Incubation with enzymes + Phenol extraction--> No DNA
• Chromatin Incubation without enzymes + Phenol extraction --> No DNA
• Chromatin Incubation with enzymes + Column extraction --> White pellet and  No DNA
• Chromatin Incubation without enzymes + Column extraction --> White pellet and No DNA
• Buffer without EDTA Incubation without enzymes --> White pellet
• Buffer with EDTA Incubation without enzymes --> White pellet

In all cases, unlike my previous extraction, the pellet appeared only after adding the binding buffer  and was easy to resuspend by adding isopropanol (sorry Nirmalya I didn’t see your message on time) or simply mixing.  But the “unresuspendible” stone-like pellet of my previous and also negative extraction (buffer with EDTA and no enzymes incubations) didn’t appeared…

Binding buffer must be yellow if pH is optimal, and it was in all cases. However, in the tubes with buffer only (no chromatin),  I add NaOAc pH5.2 to low more the pH trying to see the Stone-like pellet (theoretically a precipitation of EDTA), but didn’t happened. So it seems that what caused the appearance of that pellet, was… maybe a combination between EDTA and proteins??

So now the question is obvious; why I didn’t get DNA in this extraction? Could it have been degraded by residual nucleases in the 3h 37ºC incubation (with and without protK) ?

Further information: the chromatin I used was extracted weeks ago and have been thawed twice, but refrozen with liquid nitrogen each time.

Talat,  in the step 5

 “now suspended in 50uL of Elution buffer, add 50uL of Reverse Cross-Linking Buffer and mix well by pipeting. Separate the magnetic beads on a magnetic stand and transfer the 100uL of “Eluted chromatin” (it is still cross-linked) to a clean tube.” 

Is pipeting with these buffers sufficient to separate dynabeads from AB-chromatin without any incubation?

Eddy, I have been thinking about that option, I still have not tried because I have not seen it done in any paper I've read. But maybe it would be a good idea, doing the decrosslinking, protein degradation and beads separation. But I am concerned that the dynabeads are degraded in the process (by the prot K), contaminating my simple… It is possible?

About RNA:

Why don’t you  guys add RNase directly in the ON 65ºC incubation? And, anyway, what's wrong with having some RNA in the sample?

It should work with  DynaBeads as well. You may incubate at 37C for 15 min and then try separating the DynaBeads... the buffer supplied by Active Motif is validated to ELUTE the chromatin off the magnetic beads without any incubation but you may incubate at 37C for 15 min to aid in the elution.

Excess of RNA may compete with DNA for the binding with filter on the column. That is why it is recommended to degrade it by RNase. But normal RNase should loose its activity at 65C, its optimal temperature is 37C. However, if your buffer contains Mg2+ ions the RNA undergoes self hydrolysis at 65 C and should be completely eliminated, so if your buffer contains Mg2+ ions (and no EDTA to chelate it) there is no need to use RNase.

In addition, crosslinked complex of DNA and protein behaves as protein, so in phenol-chlorophorm extraction it goes to interphase. That is why before any procedure of DNA purification from cross-linked chromatin you should decrosslink comlexes and use proteinse K to degrade any proteins in the sample. I think nothing serious can go from dynabids to your sample only antibodies if they are coupled on the beads. All peptides from the antibodies the will be removed during DNA extraction.

Moreover, you mention that you use soluble chromatin that is in the supernatant.  Cross-linked chromatin is very  hard to solubilize by sonication. It is required 2.5 - 3 minutes at maximum power of the sonicator to get enough chromatin fragmentation. To increase chromatin solubility, it is usually used 1% SDS that is strong ionic detergent, and then should be diluted at least 10 times for further processing. You can see my protocol published in my paper attached. This protocol for yeast, but some steps is common for ChIP in other organims. May be you can spot some usefull moments in my protocol.

I have the same problem. I do not know how to do next.