no-antibody control is stronger than my sample! - in ChIP.. (Dec/11/2007 )
Hi, everybody.
It's nice of this palce's existence. I am a new guy.
I have done ChIP for a couple of weeks. At first, everything goes smoothly, but now i have met a real problem.
I am performing ChIP-chip. At first, my sonication is strong and the DNA fragments after LM-PCR is too small, but the ChIP itself is good. I use one gene that known to be bound with my protein to test the efficiency of ChIP. The sample that added antibody has a strong band and no-antibody control is none.
But the DNA fragments is too small to be well-labelled. So I lowered the strength of sonication, the results is worse. Even no-antibody control is stronger than my sample. i thought the strong background maybe because the DNA after sonication is too large. Then, I toned up the sonication strength again. But unfortunatly, no-antibody control is still stronger than added antibody sample.
I was confused! Can you tell me why?
Another important detail i want to tell you, everytime i finished ChIP, I precipitated the DNA that eluted, i can see the DNA precipitation in the bottom of the tube. is it abnormal?
Thank you for any help!
lorry
It's nice of this palce's existence. I am a new guy.
I have done ChIP for a couple of weeks. At first, everything goes smoothly, but now i have met a real problem.
I am performing ChIP-chip. At first, my sonication is strong and the DNA fragments after LM-PCR is too small, but the ChIP itself is good. I use one gene that known to be bound with my protein to test the efficiency of ChIP. The sample that added antibody has a strong band and no-antibody control is none.
But the DNA fragments is too small to be well-labelled. So I lowered the strength of sonication, the results is worse. Even no-antibody control is stronger than my sample. i thought the strong background maybe because the DNA after sonication is too large. Then, I toned up the sonication strength again. But unfortunatly, no-antibody control is still stronger than added antibody sample.
I was confused! Can you tell me why?
Another important detail i want to tell you, everytime i finished ChIP, I precipitated the DNA that eluted, i can see the DNA precipitation in the bottom of the tube. is it abnormal?
Thank you for any help!
lorry
Hi, I went to a chip-chip meeting yesterday and learnt that LM-PCR will make smaller fragments than other amplification methods. What size fragments are you getting?
Hi, Clare.
I usually got LM-PCR fragments of 100bp to 1Kb. I have tried several times, but my sonication can not avoid 100bp fragments. So my LM-PCR fragments may be a little small.
Can you share me with your understanding of ChIP-chip or LM-PCR?
In ChIP-chip, what is the most important in reducing false positive? and, how LM-PCR's cycle number affects final result?
Thank you!
lorry
I usually got LM-PCR fragments of 100bp to 1Kb. I have tried several times, but my sonication can not avoid 100bp fragments. So my LM-PCR fragments may be a little small.
Can you share me with your understanding of ChIP-chip or LM-PCR?
In ChIP-chip, what is the most important in reducing false positive? and, how LM-PCR's cycle number affects final result?
Thank you!
lorry
Hi again,
I personally do not use LM-PCR as it's tricky and so can't really comment any further. I use the WGA kit from Sigma to amplify my DNA. I do know that if I do too many amplification cycles expression of my control genes is crap via real time PCR. How exactly have you played around with sonication? There are many important factors (depending on what kind of sonicator you have) and you'll have to optimise conditions for every type of cell you use. I use a Bioruptor which gives awesome consistent results. For a cell line I use I sonicate cells at a conc, of 1 million cells/30ul and always get fragments about 300-1000 (5 mins, 30 sec on/off). But for my patient samples I have to sonicate for longer. As for the Chip-chip bit, I haven't got to the hybridisation step yet. I am still working out the best way to do things. Sorry I can't be of any more help.
Clare
I know Bioruptor. This machine is indeed ideal for ChIP sonication.
But I don't have this machine pityingly. Now how to decrease background is my biggest trouble.
Good luck with your experiments, Clare.
lorry
But I don't have this machine pityingly. Now how to decrease background is my biggest trouble.
Good luck with your experiments, Clare.
lorry
Perhaps if you post your protocol (esp. the pre-clearing step) someone could give you more advice. I know you can pre-block the beads with BSA and ssDNA - I will be trying it this week.
Clare