qPCR Results

I got the same results as I did before when I repeated the qPCR, which is good in a way because I know that I’m doing the qPCR part right. I got lower standard deviation than I did before but the % input was still about 0.1%. This means that there’s a problem with my ChAP procedure, which is bad because it’s a long procedure and there’s a lot of things that could have gone wrong. Our best guess right now is that there is an issue with the sonication step, specifically that I’m not sonicating enough. I’m supposed to check my sonication but I had to check it on a Saturday and I didn’t know how to do the check. Since no one was in lab that Saturday, I skipped thinking it wouldn’t be that important. I probably should have been more thorough in retrospect. If I don’t sonicate enough, the DNA pieces aren’t as small as we would like them to be. I’m not exactly sure why this would cause my % input to be so low but I have a couple theories. First, right before we do the qPCR, we have to do a DNA purification step to filter out any proteins or cell debris. This step filters out large pieces so if my DNA is too large in size, it could get filtered out which would mean that I wouldn’t get a lot of DNA in the sample that I run for qPCR. The other possibility is that DNA pieces of larger size might not bind to the beads as well as smaller pieces do. This would again result in less DNA being purified and cause my % input to be too low. 

To solve this problem and make sure that sonication isn’t an issue, I’m going to perform a mini experiment where I check how much sonication is required. I’m going to do everything like I normally do in my ChAP procedure up to the sonication point. At this step, I’m going to sonicate for a certain number of cycles to determine how many cycles are necessary to get small enough DNA. This should allow me to see if sonication was an issue and how much I should sonicate in the future.