Problem
|
Causes
|
Solution
|
Chromatin is under sheared and fragments are too big. |
Too many cells have been used. |
Reduce the number of cells. |
The shearing is too low. |
Increase the number of shearing cycles. |
Increase the shearing peak power, the duty factor and/or the cycles/burst. |
Chromatin is over sheared and fragments are too small. |
Too few cells have been used. |
Increase the number of cells. |
The shearing is too high. |
Reduce the number of shearing cycles. |
Reduce the shearing peak power, the duty factor and/or the cycles/burst. |
Too low recovery over IgG control and/or negative control (high background). |
Not enough chromatin used for the experiment. |
Increase the amount of chromatin. |
Amount of antibody is too low. |
Increase the amount of antibody. |
Amount of antibody is too high. |
Reduce the amount of antibody. |
The antibody has low specificity. |
Change the antibody. |
Washing conditions are too stringent. |
Reduce the number of washing. |
Reduce the stringency of the washing buffers. |
Washing conditions are not stringent enough. |
Increase the number of washing. |
Increase the stringency of the washing buffers. |
Too much DNA was pipetted in the qPCR. |
Reduce the amount of DNA pipetted in the qPCR. |
qPCR conditions are not optimal. |
Optimize the qPCR conditions. |
Reduce the number of cycles. |
No product or product is too low in the qPCR reaction of the input sample. |
Not enough DNA was pipetted in the qPCR. |
Increase the amount of DNA pipetted in the qPCR. |
qPCR conditions are not optimal. |
Optimize the qPCR conditions. |
Increase the number of cycles. |
Not enough chromatin used for the experiment. |
Increase the amount of chromatin. |
No signal in the positive control and/or panH3 ChIP. |
Not enough chromatin used for the experiment. |
Increase the amount of chromatin. |
Amount of antibody is too low. |
Increase the amount of antibody. |
The antibody has low specificity. |
Change the antibody. |
Elution is sub-optimal. |
Be sure to frequently vortex the samples to maintain the beads in solution. |