Input chip doesn't work! - (Dec/14/2010 )
Hi all,
I am pretty new with chromatin immunoprecipitation and I'd like to ask for some help!
I am using the magna chip from millipore and the input doesn't work. I am following the protocol removing the input after the sonication but when I do the pcr the input doesn't show any band while I see band in the treated samples! I also did a control pcr using primer for gapdh and I obtained a good band for the positive control (anti-RNA polymerase II) but not for the input!
Any clue? I am totally lost!
Thank you.
-milla-
milla on Tue Dec 14 23:32:43 2010 said:
Hi all,
I am pretty new with chromatin immunoprecipitation and I'd like to ask for some help!
I am using the magna chip from millipore and the input doesn't work. I am following the protocol removing the input after the sonication but when I do the pcr the input doesn't show any band while I see band in the treated samples! I also did a control pcr using primer for gapdh and I obtained a good band for the positive control (anti-RNA polymerase II) but not for the input!
Any clue? I am totally lost!
Thank you.
This may be a stupid question but, are you treating the input similarly to how you treat the IPed samples after they're eluted off the beads (i.e. the same proteinase K treatment, reversal of crosslinking, and DNA isolation)?
-KPDE-
Yes,
The Input is removed after sonication and then included again in the process after the washes of the beads (treated with proteinase K and elution buffer followed by DNA purification using spin column).
This may be a stupid question but, are you treating the input similarly to how you treat the IPed samples after they're eluted off the beads (i.e. the same proteinase K treatment, reversal of crosslinking, and DNA isolation)?
-milla-
milla on Wed Dec 15 16:29:02 2010 said:
Yes,
The Input is removed after sonication and then included again in the process after the washes of the beads (treated with proteinase K and elution buffer followed by DNA purification using spin column).
This may be a stupid question but, are you treating the input similarly to how you treat the IPed samples after they're eluted off the beads (i.e. the same proteinase K treatment, reversal of crosslinking, and DNA isolation)?
Okay, another stupid question. Is it possible that the amount of input you are using is too much for the spin column (i.e. could it be clogging the filter?)
-KPDE-
The amount of input removed after the sonication is 5 ul (1%).
Meanwhile I also read the samples with the nanodrop (I am not sure if make sense but I was curious!)... all the sample (real+input) have really low concentration (3-10 ng/ul) and bad absorbance but inputs are the worse with even a negative A260/280!!
No problem for the "stupid" questions... they may be stupid for you but not for a new ChIP user like me!
Thank you!
-milla-
Have you tried running any of the input on an agarose gel to see what the shearing looks like? This will also give you an idea of how much DNA is in there.
-ezilybored-
ezilybored on Thu Dec 16 10:24:51 2010 said:
Have you tried running any of the input on an agarose gel to see what the shearing looks like? This will also give you an idea of how much DNA is in there.
The run of the sonicated samples looks ok, but the run of the input DNA (at the end of the protocol) doesn't show anything!
-milla-
milla on Thu Dec 16 21:10:38 2010 said:
ezilybored on Thu Dec 16 10:24:51 2010 said:
Have you tried running any of the input on an agarose gel to see what the shearing looks like? This will also give you an idea of how much DNA is in there.
The run of the sonicated samples looks ok, but the run of the input DNA (at the end of the protocol) doesn't show anything!
What's your DNA isolation protocol?
-KPDE-
