Input problem with fast chip protocol - (Apr/22/2008 )
Hello everybody,
I finally got good results with ChIp against transcription factors, but I used a lot of chromatin (150mg of fresh liver). But my problem is that my input has a high Ct (30), which is the same as the ChIp sample. I don't understand because after ethanol precipitation I can see a big pellet. When I dilute my input, there is no difference in Ct ( I thought that I had too much material). Does anyone have an idea to solve my problem?
I have an other problem, I tried to use magnetic beads (ademtech) to have less background, but I got low Ct in IgG samples, did someone had good results using magnetic beads and chelex ?
Thank for your help
I finally got good results with ChIp against transcription factors, but I used a lot of chromatin (150mg of fresh liver). But my problem is that my input has a high Ct (30), which is the same as the ChIp sample. I don't understand because after ethanol precipitation I can see a big pellet. When I dilute my input, there is no difference in Ct ( I thought that I had too much material). Does anyone have an idea to solve my problem?
I have an other problem, I tried to use magnetic beads (ademtech) to have less background, but I got low Ct in IgG samples, did someone had good results using magnetic beads and chelex ?
Thank for your help
Is it possible that something in your input is inhibitory to PCR?
As for the low Ct in your IgG sample, was this higher or lower than you were expecting. If you are trying to eliminate any pull down at all with IgG you may need to try using a blocking reagent (e.g. 5% BSA/100ug/ml salmon sperm DNA).
I finally got good results with ChIp against transcription factors, but I used a lot of chromatin (150mg of fresh liver). But my problem is that my input has a high Ct (30), which is the same as the ChIp sample. I don't understand because after ethanol precipitation I can see a big pellet. When I dilute my input, there is no difference in Ct ( I thought that I had too much material). Does anyone have an idea to solve my problem?
I have an other problem, I tried to use magnetic beads (ademtech) to have less background, but I got low Ct in IgG samples, did someone had good results using magnetic beads and chelex ?
Thank for your help
Is it possible that something in your input is inhibitory to PCR?
As for the low Ct in your IgG sample, was this higher or lower than you were expecting. If you are trying to eliminate any pull down at all with IgG you may need to try using a blocking reagent (e.g. 5% BSA/100ug/ml salmon sperm DNA).
Thank you for your answer
I don't think that someting could inhibit PCR in the input because I use the buffer of your protocol and the results are reproducible, so it's not due to chelex contamination.
The low Ct for IgG occurs only with the magnetic beads which are provided with a blocking buffer; but I think that I'm gonna try other magnetic beads or stay with agarose beads
I finally got good results with ChIp against transcription factors, but I used a lot of chromatin (150mg of fresh liver). But my problem is that my input has a high Ct (30), which is the same as the ChIp sample. I don't understand because after ethanol precipitation I can see a big pellet. When I dilute my input, there is no difference in Ct ( I thought that I had too much material). Does anyone have an idea to solve my problem?
I have an other problem, I tried to use magnetic beads (ademtech) to have less background, but I got low Ct in IgG samples, did someone had good results using magnetic beads and chelex ?
Thank for your help
Is it possible that something in your input is inhibitory to PCR?
As for the low Ct in your IgG sample, was this higher or lower than you were expecting. If you are trying to eliminate any pull down at all with IgG you may need to try using a blocking reagent (e.g. 5% BSA/100ug/ml salmon sperm DNA).
Thank you for your answer
I don't think that someting could inhibit PCR in the input because I use the buffer of your protocol and the results are reproducible, so it's not due to chelex contamination.
The low Ct for IgG occurs only with the magnetic beads which are provided with a blocking buffer; but I think that I'm gonna try other magnetic beads or stay with agarose beads
To give you an idea about what dilution I use for the input, I take about 10ul of chromatin (about 100,000 cells more or less) and, after extracting the DNA as per the protocol, I dilute about 40X before running on PCR. For most of my primers at this dilution, I get a Ct in the mid 20s. If you are having trouble getting lower Cts one possible problem is the redissolving of the pellet after EtOH precipitation. I usually have to dry the pellet to complete dryness (when it becomes transparent) to get it to dissolve, even after boiling.
As for the beads, are you able to get no amplification using agarose beads? If so, let me know what you're doing because I always get a significant background using agarose. It hasn't been a problem for ChIP on histones, histone mods, or Pol II but for some things that bind at low level, the background can be problematic.
To give you an idea about what dilution I use for the input, I take about 10ul of chromatin (about 100,000 cells more or less) and, after extracting the DNA as per the protocol, I dilute about 40X before running on PCR. For most of my primers at this dilution, I get a Ct in the mid 20s. If you are having trouble getting lower Cts one possible problem is the redissolving of the pellet after EtOH precipitation. I usually have to dry the pellet to complete dryness (when it becomes transparent) to get it to dissolve, even after boiling.
As for the beads, are you able to get no amplification using agarose beads? If so, let me know what you're doing because I always get a significant background using agarose. It hasn't been a problem for ChIP on histones, histone mods, or Pol II but for some things that bind at low level, the background can be problematic.
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I will try to dry completely my pellet and see if it gives lower Ct.
I have bacground using agarose but at least there is a difference between antibody and IgG. I get 29ct for my antibody and >33ct for IgG. The antibody I use is against a TF and I use 120mg of fresh tissue by Ip!
I dryed my pellet before resuspending in chelex but there is no improvement with QPCR.
I have another problem with the fast chip protocol. Using this protocol I never could see my DNA on agarose gel. After reverse cross link (using chelex or traditionnal reverse cross link), there is no DNA on my gel!
The only thing I can see is, before reverse cross link a high weight band. Do you think that my DNA is not sheared?
But how could I obtain good results with QPCR if my DNA is not sheared.
I sonicated DNA without cross linking and I could see a nice smear between 600bp and1kb.
I have another problem with the fast chip protocol. Using this protocol I never could see my DNA on agarose gel. After reverse cross link (using chelex or traditionnal reverse cross link), there is no DNA on my gel!
The only thing I can see is, before reverse cross link a high weight band. Do you think that my DNA is not sheared?
But how could I obtain good results with QPCR if my DNA is not sheared.
I sonicated DNA without cross linking and I could see a nice smear between 600bp and1kb.
Were you able to see that your pellet dissolved completely after the boiling. I keep wondering if your DNA is getting lost with the pellet not dissolving. One thing you might try is adding the 100ul of chelex directly to your chromatin aliquot (no EtOH precipitation). Boil and treat with prot K (there shouldn't be very much protease inhibitor left after the boiling unless you are using a very stable cocktail). Then extract the DNA from the supernatant using a Qiagen PCR cleanup or enzyme reaction cleanup kit (or something similar). Sorry this is being such a pain for you. The idea was for this method to be easier than the traditional one.