no PCR product from input... - (Jul/09/2010 )
I am new to this forum and new to ChIP...
two weeks ago, i did a ChIP with my boss
the result showed no PCR product from the ChIP, but input does
it was with 1 well of ES cells from a 6-well plate, using bioruptor for sonication, 1ml solution
then I went on to repeating it, but this time i used 3 wells of cells, in 2 ml of solution.
I ran the lysate on 1% agarose, both showed a similar profile, but of course mine is more intense due to more starting material.
i then decrosslink by adding 4ul 5M NaCl to 100ul diluted lysate, 95C, 20 min (i tried 15min as well)
then went onto do PCR with the input.
Input from the 1st experiment showed PCR product, but the 2nd time didn't...
so what would be the problem?
am i started with to many cells?
or if it is the problem with the decrosslinking?
thanks very much
I don't think more starting material is the problem. How did you purify your DNA after reverse crosslinking? You might have lost your DNA, or have impurities in your DNA that inhibited PCR reaction.
crono on Jul 9 2010, 02:40 PM said:
I'm surprised you got any usable DNA the first time with that protocol. In my experience, if you heat the DNA to 95C for 15-20 min without either using a chelating agent (1mM EDTA or 10% chelex), raising the pH above 9.8, or both, then the DNA is of poor quality as a template. Also, there's no need for 200mM NaCl if you are heating to 95C. I'm not exactly sure what the presence of NaCl accomplishes (maybe nucleophilic substitution of the methylene bridge?? No idea actually) however I've only seen it used in the protocol for reversal of crosslinking at lower temps (i.e. 65C) and also in the presence of bicarbonate.
I purify the DNA (ChIP) product with phenol:chloroform extraction then EtOH ppt.
but not the input, i just diluted the lysate, heated it then PCR...
really have no idea what's wrong...
will try another batch of cells again this week...
So you did not revser crosslink your input DNA before PCR? You have to at least reverse crosslink the input DNA.
I heated it to 95C for 20 minutes...
really have no idea what's wrong...
unless there are some inhibitors in the lysis buffer...
but I did it according to the Upstate kit's protocol and diluted the lysate 10 folds with the dilution buffer...
For what it's worth I briefly ran into an issue with not getting amplification of my input DNA. I ran 9 samples, and for some odd reason the input from 5 of them did not amplify; I concluded that it had to be a PCR inhibitor, as I got good looking amplification from my IP'd samples. To solve the problem I was just a bit more thorough in my purification of my input samples. I use the QiaQuick kit, so I just make sure to vortex my input samples with the PB buffer provided instead of adding my input to the PB buffer. Haven't had the problem since...
reverse crosslinking is a very crucial step in the ChIP protocol.
First of all, you have to check the sonication efficiency before you start the whole ChIP procedure. In this way you will already see if your DNA is okay or not. In this case a short crosslinking procedure is fine. I am doing:
addition of 2 ul of 5 M NaCl to 50 ul of the samples and boil it for 15 min. After that it is cooled down to room temperature and 1 ul of 1 ug/ml RNase A are added for 5 min, and then 0.5 ul of 20 mg/ml proteinase K are added for 30 min at 55 degrees.
This is then purified with Phenol-Chloroform (any other method of DNA purification will not work).
and the DNA is checked on a 1% agarose gel. Fragments should be between 500 and 1500 bp.
Reverse Crosslinking after the CHIP I do as follows:
addition of 2 ul 5M NaCl to 200 ul of the ChIP samples, input samples are filled up with elution buffer
4 h incubation at 65degrees
after that addition of 1 ul 10 mg/ml RNase A for 30 min at 37degrees celsius
and then 1 ul of 20 mg/ml Proteinase K over night at 55 degrees
Precipitation of DNA I usually do overnight at -20 degrees, it increases the yield of DNA in 100% ethanol and Sodiumacetate.
Not proper removal of proteins from the DNA will interfere with the real time PCR.
i tried it again and it didn't work...
so i tried to do a phenol:chloroform extraction with my input... but no luck...
i am working on a new batch of cell now and will try to use Proteinase K as well...
and i have another question.
when I sonicate the sample with Bioruptor, I stop and have a look at my samples and mix the ice for every 2 pulses.
I found that the solutions become very milky sometime...
I wonder if this is normal?
cant really tell if it is the SDS precipitated out or the samples were boiling and those are actually small bubbles...,
but everything was cold when i check it