ChIP HELP!! - (May/15/2009 )
Hi, I am doing ChIP assay on MCF-7 cells immunoprecipitating P53 and HIF-1a protein-DNA crosslinks. I have tried it 4 times. The first time, for unknown reasons, I get pure usable DNA from the immunoprecipitation (even without doing extra DNA purification process). However, I can't repeat my results afterwards. I keep getting heavily contaminated samples. The 260/280 reading is like 0.5 and 260/230 is like 0.15. These samples I tried to do RT-PCR with and it just doesnt work since its too dirty.. Then I tried to purify the DNA with Qiagen PCR Purification Kit and Phenol Chloroform. Both the methods completely take out all my DNA since there isn't much DNA to start with (After the immunoprecipitation I get like 50-70ng/ul of DNA). So for those who have done ChIP before. Do you guys need to do extra purification process? And how you guys do it? If you dont need to do it... please tell me something that I can improve on... so i wont get samples thats so contaminated. Cheers!
-Travis-
Travis on May 15 2009, 01:02 PM said:
Hi, I am doing ChIP assay on MCF-7 cells immunoprecipitating P53 and HIF-1a protein-DNA crosslinks. I have tried it 4 times. The first time, for unknown reasons, I get pure usable DNA from the immunoprecipitation (even without doing extra DNA purification process). However, I can't repeat my results afterwards. I keep getting heavily contaminated samples. The 260/280 reading is like 0.5 and 260/230 is like 0.15. These samples I tried to do RT-PCR with and it just doesnt work since its too dirty.. Then I tried to purify the DNA with Qiagen PCR Purification Kit and Phenol Chloroform. Both the methods completely take out all my DNA since there isn't much DNA to start with (After the immunoprecipitation I get like 50-70ng/ul of DNA). So for those who have done ChIP before. Do you guys need to do extra purification process? And how you guys do it? If you dont need to do it... please tell me something that I can improve on... so i wont get samples thats so contaminated. Cheers!
Hi there
Do you want to send me your ChIP protocol to see if I can spot anything that may be causing you problems? We purify our ChIP DNA using Qiagen PCR Purification Kit only (use buffer PB as the binding buffer). We don't need to purify any further
Clare
-Clare-
Travis on May 15 2009, 05:02 AM said:
Hi, I am doing ChIP assay on MCF-7 cells immunoprecipitating P53 and HIF-1a protein-DNA crosslinks. I have tried it 4 times. The first time, for unknown reasons, I get pure usable DNA from the immunoprecipitation (even without doing extra DNA purification process). However, I can't repeat my results afterwards. I keep getting heavily contaminated samples. The 260/280 reading is like 0.5 and 260/230 is like 0.15. These samples I tried to do RT-PCR with and it just doesnt work since its too dirty.. Then I tried to purify the DNA with Qiagen PCR Purification Kit and Phenol Chloroform. Both the methods completely take out all my DNA since there isn't much DNA to start with (After the immunoprecipitation I get like 50-70ng/ul of DNA). So for those who have done ChIP before. Do you guys need to do extra purification process? And how you guys do it? If you dont need to do it... please tell me something that I can improve on... so i wont get samples thats so contaminated. Cheers!
If you are using an SDS buffer to elute your complexes and if you are using proteinase K you will need to do some kind of purification. You might try the mini-elute kit or equivalent so that you don't lose the small amount of DNA that you have.
On the other hand, a technique that we use is to add 10% chelex suspension or a high pH buffer (25mM Tris, 1mM EDTA; no need to adjust pH as it should be around pH10) along with 20µg of proteinase K directly to your protein A beads after the last wash. Digest at 55C for 15 minutes and boil (100C) for 10-15 minutes. If you use the chelex, just transfer the supernatant and wash the chelex/beads with the same volume and pool with the previous supernatant. The DNA at this point is ready for PCR (as the high temp elutes your DNA, kills the proteinase K, and reverses the crosslinks, simultaneously). If you use the high pH Tris, you can use the DNA directly in PCR (the advantage of the high pH tris is that your DNA remains stable for quite a long time). We've published this method (using the chelex) and it has worked for quite a number of labs (80+ citations since 2006) so it will likely work for you as well. If you would like a protocol I can send one.
-KPDE-
I am using the same protocol i think. I reverse the crosslink with 10% chelex beads by boiling in 95c for 10 minutes. Then add in proteinase K. Is it possible that the heating process can't reverse the crosslink efficiently? or the proteinase K isnt efficient in digesting the protein? Coz my samples always have a very low 260/280 value. If thats the case, anything i can do to remove the protein in the samples?
KPDE on May 15 2009, 12:37 PM said:
Travis on May 15 2009, 05:02 AM said:
Hi, I am doing ChIP assay on MCF-7 cells immunoprecipitating P53 and HIF-1a protein-DNA crosslinks. I have tried it 4 times. The first time, for unknown reasons, I get pure usable DNA from the immunoprecipitation (even without doing extra DNA purification process). However, I can't repeat my results afterwards. I keep getting heavily contaminated samples. The 260/280 reading is like 0.5 and 260/230 is like 0.15. These samples I tried to do RT-PCR with and it just doesnt work since its too dirty.. Then I tried to purify the DNA with Qiagen PCR Purification Kit and Phenol Chloroform. Both the methods completely take out all my DNA since there isn't much DNA to start with (After the immunoprecipitation I get like 50-70ng/ul of DNA). So for those who have done ChIP before. Do you guys need to do extra purification process? And how you guys do it? If you dont need to do it... please tell me something that I can improve on... so i wont get samples thats so contaminated. Cheers!
If you are using an SDS buffer to elute your complexes and if you are using proteinase K you will need to do some kind of purification. You might try the mini-elute kit or equivalent so that you don't lose the small amount of DNA that you have.
On the other hand, a technique that we use is to add 10% chelex suspension or a high pH buffer (25mM Tris, 1mM EDTA; no need to adjust pH as it should be around pH10) along with 20µg of proteinase K directly to your protein A beads after the last wash. Digest at 55C for 15 minutes and boil (100C) for 10-15 minutes. If you use the chelex, just transfer the supernatant and wash the chelex/beads with the same volume and pool with the previous supernatant. The DNA at this point is ready for PCR (as the high temp elutes your DNA, kills the proteinase K, and reverses the crosslinks, simultaneously). If you use the high pH Tris, you can use the DNA directly in PCR (the advantage of the high pH tris is that your DNA remains stable for quite a long time). We've published this method (using the chelex) and it has worked for quite a number of labs (80+ citations since 2006) so it will likely work for you as well. If you would like a protocol I can send one.
-Travis-
Travis on May 18 2009, 08:26 AM said:
I am using the same protocol i think. I reverse the crosslink with 10% chelex beads by boiling in 95c for 10 minutes. Then add in proteinase K. Is it possible that the heating process can't reverse the crosslink efficiently? or the proteinase K isnt efficient in digesting the protein? Coz my samples always have a very low 260/280 value. If thats the case, anything i can do to remove the protein in the samples?
If you are using an SDS buffer to elute your complexes and if you are using proteinase K you will need to do some kind of purification. You might try the mini-elute kit or equivalent so that you don't lose the small amount of DNA that you have.
On the other hand, a technique that we use is to add 10% chelex suspension or a high pH buffer (25mM Tris, 1mM EDTA; no need to adjust pH as it should be around pH10) along with 20µg of proteinase K directly to your protein A beads after the last wash. Digest at 55C for 15 minutes and boil (100C) for 10-15 minutes. If you use the chelex, just transfer the supernatant and wash the chelex/beads with the same volume and pool with the previous supernatant. The DNA at this point is ready for PCR (as the high temp elutes your DNA, kills the proteinase K, and reverses the crosslinks, simultaneously). If you use the high pH Tris, you can use the DNA directly in PCR (the advantage of the high pH tris is that your DNA remains stable for quite a long time). We've published this method (using the chelex) and it has worked for quite a number of labs (80+ citations since 2006) so it will likely work for you as well. If you would like a protocol I can send one.
KPDE on May 15 2009, 12:37 PM said:
Travis on May 15 2009, 05:02 AM said:
Hi, I am doing ChIP assay on MCF-7 cells immunoprecipitating P53 and HIF-1a protein-DNA crosslinks. I have tried it 4 times. The first time, for unknown reasons, I get pure usable DNA from the immunoprecipitation (even without doing extra DNA purification process). However, I can't repeat my results afterwards. I keep getting heavily contaminated samples. The 260/280 reading is like 0.5 and 260/230 is like 0.15. These samples I tried to do RT-PCR with and it just doesnt work since its too dirty.. Then I tried to purify the DNA with Qiagen PCR Purification Kit and Phenol Chloroform. Both the methods completely take out all my DNA since there isn't much DNA to start with (After the immunoprecipitation I get like 50-70ng/ul of DNA). So for those who have done ChIP before. Do you guys need to do extra purification process? And how you guys do it? If you dont need to do it... please tell me something that I can improve on... so i wont get samples thats so contaminated. Cheers!
If you are using an SDS buffer to elute your complexes and if you are using proteinase K you will need to do some kind of purification. You might try the mini-elute kit or equivalent so that you don't lose the small amount of DNA that you have.
On the other hand, a technique that we use is to add 10% chelex suspension or a high pH buffer (25mM Tris, 1mM EDTA; no need to adjust pH as it should be around pH10) along with 20µg of proteinase K directly to your protein A beads after the last wash. Digest at 55C for 15 minutes and boil (100C) for 10-15 minutes. If you use the chelex, just transfer the supernatant and wash the chelex/beads with the same volume and pool with the previous supernatant. The DNA at this point is ready for PCR (as the high temp elutes your DNA, kills the proteinase K, and reverses the crosslinks, simultaneously). If you use the high pH Tris, you can use the DNA directly in PCR (the advantage of the high pH tris is that your DNA remains stable for quite a long time). We've published this method (using the chelex) and it has worked for quite a number of labs (80+ citations since 2006) so it will likely work for you as well. If you would like a protocol I can send one.
You'll have the low 260/280 ratio because Fast ChIP does not remove the resulting peptides/amino acids from the proteinase K digestion. If you are only doing PCR or qPCR there is no need to remove them. For instance, I typically do PCR with SYBR green and my ChIP templates often have a 260-280 ratio closer to 1 than to 1.7. I have no problem getting consistent results and 94-100% amplification efficiencies.
-KPDE-
Ok so I dont need very pure DNA from ChIP. But then I am getting like 0.4 in 260/280. I tried running q-PCR with those samples and they just dont work... so I just thought I should reduce the contamination alittle
-Travis-