2 replies to this topic

[doudou30]

Hi !  

I have just quantified with agilent chip the size and quantity of fragments I have immunoprecipitated and I observe 2 smears, one at 100/250 pb and one at 1000/10000 pb. After sonication I have only a smear around 200bp (on agarose gel). So I wonder what is this second smear. Is it agregated chromatine ? A problem with sonication ? Bad reverse cross-link ? ...Or is it normal ?
I just want to be sure it'll not be a problem for my library preparation (concentration of adaptater for exemple which could bind these bigger fragments).  

Thanks for your help !!

[KPDE]

doudou30, on May 6 2010, 03:28 PM, said:

Hi !  

I have just quantified with agilent chip the size and quantity of fragments I have immunoprecipitated and I observe 2 smears, one at 100/250 pb and one at 1000/10000 pb. After sonication I have only a smear around 200bp (on agarose gel). So I wonder what is this second smear. Is it agregated chromatine ? A problem with sonication ? Bad reverse cross-link ? ...Or is it normal ?
I just want to be sure it'll not be a problem for my library preparation (concentration of adaptater for exemple which could bind these bigger fragments).  

Thanks for your help !!

Unless you RNAse treated your samples, that second smear you see before sonication is probably RNA.

[doudou30]

thanks for your answer
In fact I found the solution ! It is not RNA (I have treated my samples). In fact the bigger fragments (1000/10000) are concentrated in a very small portion of the agilent chip and because I have initially a smear a DNA (even If I have more DNA at around 200bp), these bigger fragments are grouped together on the chip and formed this bigger smear. I don't no if I am clear, but the conclusion is that everything is ok  
But thanks for you help anyway !