Low DNA concentration for sequencing - (May/28/2013 )

Hi all,
I have been setting up the ChIP protocol for some nuclear receptors from mouse liver and my idea was to send the samples for sequencing. But I am quite frustrated because even if I get a nice enrichment over the IgG (between 6 and 9 cycles, Ct values around 21-24), I am not able to quantify the DNA in the Qubit. Some people told me that starting from 200 mg of liver they get around 5-10 ng of DNA, so I don´t know where I am loosing my DNA. Last time I tried increasing the magnetic beads to 100ul per ChIP, and using 50 mg of liver (300ul sonicate) I could get around 2 ng (I was so happy!). But when I scale up and set up 10 tubes with 200 mg and 400ul beads each, and then pool the elutes and concentrate them, I got only 6ng in total (I would expect around 80)l!!!! I am using the QIAquick columns from QIAGEN, and I recently order the MinElute PCR purification kit, because I can elute the samples in a smaller volume. But I don´t know what my problem can be. I do the reverse crosslinking at 65C over night, and then treatment with RNase, 30min at 37C and proteinase K, 1h at 55C. Do you think I might be loosing the sample in the column? or the amount of beads maybe is interfering with the immunoprecipitation? I need quite a lot of sample, as we are going to send the samples to a company and they ask me around 100 ng per sample!! I would be very grateful if anyone can give me any idea. Thanks a lot.

Txaio83 on Tue May 28 08:42:43 2013 said:

Hi all,
I have been setting up the ChIP protocol for some nuclear receptors from mouse liver and my idea was to send the samples for sequencing. But I am quite frustrated because even if I get a nice enrichment over the IgG (between 6 and 9 cycles, Ct values around 21-24), I am not able to quantify the DNA in the Qubit. Some people told me that starting from 200 mg of liver they get around 5-10 ng of DNA, so I don´t know where I am loosing my DNA. Last time I tried increasing the magnetic beads to 100ul per ChIP, and using 50 mg of liver (300ul sonicate) I could get around 2 ng (I was so happy!). But when I scale up and set up 10 tubes with 200 mg and 400ul beads each, and then pool the elutes and concentrate them, I got only 6ng in total (I would expect around 80)l!!!! I am using the QIAquick columns from QIAGEN, and I recently order the MinElute PCR purification kit, because I can elute the samples in a smaller volume. But I don´t know what my problem can be. I do the reverse crosslinking at 65C over night, and then treatment with RNase, 30min at 37C and proteinase K, 1h at 55C. Do you think I might be loosing the sample in the column? or the amount of beads maybe is interfering with the immunoprecipitation? I need quite a lot of sample, as we are going to send the samples to a company and they ask me around 100 ng per sample!! I would be very grateful if anyone can give me any idea. Thanks a lot.

Hi Pito, yes, I am using RNAse before the PK treatment and I am using liver tissue as an starting material. I will follow your suggestions and try incubating more time with the PK and the phenol chloroform extraction. I also realized that my elution buffer doesn´t contain any sodium bicarbonate, and I saw that many protocols actually include this compound. I don´t know if this can make a big difference or not, but I will definitely try it. Do you have any experience with this? (I am using magnetic beads by the way). Thanks a lot!

Txaio83 on Thu Jun 13 08:36:13 2013 said:

Hi, I realize I am mixing many things in my questions, so it´s not easy to follow. Sorry! I am doing ChIP experiment from liver tissue, and so I am interested in collecting all DNA that is bound to my protein of interest. After the immunoprecipitation, the elution buffer that I am using to elute the antibody+ protein + chromatin from the magnetic beads is 50mM Tris-Hcl pH=8, 10mM EDTA and 1% SDS, O/N, at 65C. But I have seen many protocols where they use 1% SDS and 0´1M NaHCO3 for 30 minutes, they discard the beads and they continue with the reverse crosslinking, just with the eluate. As in my case I didn´t get enough material, I was thinking that maybe my elution buffer is not very strong and maybe I am loosing some DNA bound to beads. And for the DNA purification on the Qiagen kit I elute the samples with mQ water.Thank you very much!

Txaio83 on Thu Jun 13 09:30:57 2013 said:

Hi, I realize I am mixing many things in my questions, so it´s not easy to follow. Sorry! I am doing ChIP experiment from liver tissue, and so I am interested in collecting all DNA that is bound to my protein of interest. After the immunoprecipitation, the elution buffer that I am using to elute the antibody+ protein + chromatin from the magnetic beads is 50mM Tris-Hcl pH=8, 10mM EDTA and 1% SDS, O/N, at 65C. But I have seen many protocols where they use 1% SDS and 0´1M NaHCO3 for 30 minutes, they discard the beads and they continue with the reverse crosslinking, just with the eluate. As in my case I didn´t get enough material, I was thinking that maybe my elution buffer is not very strong and maybe I am loosing some DNA bound to beads. And for the DNA purification on the Qiagen kit I elute the samples with mQ water.Thank you very much!