Hi Friends,
I have done ChIP using various antibodies (histone modification, DNA methylation and TF) using Epigentek kit. After ChIP, quantified the DNA with Nanodrop and found good amount of DNA concentration (6ng- 80ng) with 260/280 around 1.5- 1.7. Since nanodrop may not be reliable, I did gel electrophorosis and found 50-60% of samples (out of 48 number) with visible DNA. Based on the these results, confidently I packed the samples for the sequencing. However, based on the Qbit results, company is saying that DNA is very low (less than 10 ng) and many samples are showing zero . I am confused with these observations, could not believe with the electrophorosis results. Now I could not make any decision whether I can proceed further or not. If you know, let me know, what is the minimum concentration of DNA for ChIP-Seq? Why there is a disparity between gel and Q bit?
Gopal, I have a Qubit instrument in my lab and I can tell you that it is very accurate. We have verified this using loading controls on real-time PCR and agarose gel electrophoresis. Might they be able to amplify the DNA in the samples that do have DNA?
Ashu, thanks for the suggestions. I have done PCR for one of the antibodies, could see good amplification. But still, I am reluctant to proceed further after hearing such low amount of DNA, will be repeating the experiments with some modifications. I was told that it is all your's plan of experiments. Great, after reading the proposal, amazed the volume of the proposed work. I wish you to have sustainable success in your scientific endeavor. Best Gopal
It is a good decision to repeat the ChIP as a low yield may cause loss of data by not capturing all targets. Glad to hear that this project is moving along. Good luck!
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