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Low DNA concentration for sequencing

chipseq DNA concentration chip elution

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#1 Txaio83

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Posted 28 May 2013 - 12:42 AM

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!!!! Posted Image 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!! Posted Image I would be very grateful if anyone can give me any idea. Thanks a lot.

#2 pito

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Posted 28 May 2013 - 04:24 AM

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!!!! Posted Image 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!! Posted Image I would be very grateful if anyone can give me any idea. Thanks a lot.

I am not sure I understand you.
You use Rnase before the proteinase treatment?
And you so start with liver for the DNA extraction?
I would incubate longer with proteinase K.. (4-5 hours or longer).
Also: try phenol chloroform extraction... Maybe the kits are getting clogged (those membranes can get clogged) + if using ethanol durin the DNA extraction in those kits, its really important the membrane is dry before adding the elution buffer (if not dried completely: the ethanol will keep the DNA on the membranes)

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 Txaio83

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Posted 13 June 2013 - 12:36 AM

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!

#4 pito

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Posted 13 June 2013 - 12:40 AM

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!


Does the protocol state you need to do RNAse before the PK treatment?

The elution buffer does not need to contain sodium bicarbonate, often people even elute with mQ water.

But just to be sure, you objective is to collect DNA from liver samples, right??

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#5 Txaio83

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Posted 13 June 2013 - 01:30 AM

Hi, I realize I am mixing many things in my questions, so it´s not easy to follow. Posted Image 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!

#6 pito

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Posted 13 June 2013 - 09:15 AM

Hi, I realize I am mixing many things in my questions, so it´s not easy to follow. Posted Image 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!

You could try it..
I am not really familiar with those protocols.

I was more talking about the steps after you got your tissue to get the DNA.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.






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