4 replies to this topic

[meatpief]

Hi All,
Im having some problems with my ChIP. We recently started nanodroping the DNA after ChIP and I have noticed massive protein contamination in my samples (as well as relatively low yields). We purify through Qiagen columns at the end of the protocol so Im unsure why there appears to be protein in the sample.
Does anyone have any ideas?
I have thought perhaps the proteinase K step isnt working or the columns are being overloaded or something. Also There are PIC and PMSF in the buffer when Proteinase K is added..wouldnt these inhibit Prot K? Seems to be in every protocol Ive looked at though. Any suggestions would be great
Thanks!

[meatpief]

meatpief, on 22 February 2011 - 03:38 AM, said:

Hi All,
Im having some problems with my ChIP. We recently started nanodroping the DNA after ChIP and I have noticed massive protein contamination in my samples (as well as relatively low yields). We purify through Qiagen columns at the end of the protocol so Im unsure why there appears to be protein in the sample.
Does anyone have any ideas?
I have thought perhaps the proteinase K step isnt working or the columns are being overloaded or something. Also There are PIC and PMSF in the buffer when Proteinase K is added..wouldnt these inhibit Prot K? Seems to be in every protocol Ive looked at though. Any suggestions would be great
Thanks!

PS Ignore the bit about PMSF etc obviously I dont have these in my elution buffer Doh!

[chabraha]

Just a thought, but when your DNA concentrations are really low (as they usually are in ChIPs) you cant really put much confidence in the contamination values that are determined by 260/280 or using the 230 reading. Also in your washes/elution why are you adding PIC? I really don't think its necessary. Have you run your sheared DNA on a gel yet to see if your ProK is good to go?

[meatpief]

chabraha, on 22 February 2011 - 09:35 AM, said:

Just a thought, but when your DNA concentrations are really low (as they usually are in ChIPs) you cant really put much confidence in the contamination values that are determined by 260/280 or using the 230 reading. Also in your washes/elution why are you adding PIC? I really don't think its necessary. Have you run your sheared DNA on a gel yet to see if your ProK is good to go?

Hi Thanks for the reply..Im not adding PIC in my wash/elution buffer I was just getting confused. Brain overload!   

I have run the sheared DNA on gel to check my digested fragment ranges is that what you mean? Sorry I'm pretty new to all of this so I'm not sure what you mean by the last bit. Just this morning I have had the same problem and the peak curves coming off the nanodrop look just like protein. However yesterday I was optimizing fixation times and checking to see if the Prot K I was using was working (I used a fresh one) and it would appear that I did not have contamination in my newer samples so I'm hoping the Prot K I have been using was just bad.

Im going to re-digest the samples I analyzed this morning with the newer Prot K and re purify and hope that works but anything you can tell me is a massive help!

Much thanks  

[chabraha]

what is your actual DNA concentration? Have you tried another method for DNA quantification, like PicoGreen? When you don't have a high enough DNA concentration you cannot trust the nano-drop readings, this is why for most experiments involving low amounts of nucleic acids bio-analyzer chips are used to determine the quality and quantity of the nucleic acid.