Yeast ChIP Help - No DNA after sonication - - (Aug/02/2007 )

Hi,

This is my first ChIP experiment, and one of my first cell-based experiments, so I am a novice and any help would be appreciated. I am trying to do a ChIP on yeast and can't find any DNA. I am using 2e7 cells, crosslinked with 1% formaldahye for 10 mins, quenched with glycine for 5 mins. To break the cell walls, I treated the cells with lyticase for 30 mins and verified under the microscope that the cells become spherical, so that the cell wall is broken. After removing the lyticase and washing the pellet, I am sonicating in 400 uL lysis buffer (50 mM Hepes, ph 7.5, 140 mM NaCl, 1 mM EDTA, 1%TritonX, 0.1% SDS, 0.1% Na Deoxycholate + PMSF). The sonicator is a Branson 450 with a cup horn, with sonication at 100% duty and output power of 5 for 5 X 30 sec with 1 min on ice between cycles. After decrosslinking with RNAase at 65C for 4 hours, I have been purifying the samples with an Invitrogen PureLink PCR kit (phenyl:chloroform is on order). When I take a UV spectra (or run on an agarose gel) there is no A260, suggesting that I have no DNA. Does anyone have an idea of where the DNA is? The postings to this site are very helpful, but focused on how to get fragments the correct size.

Thanks,
Stephanie

A fellow yeastie! Before i read your post I was going to say "did you break up the cell walls" lol.. your protocol looks mostly right by me - although we do 12 cycles for our sonications (different machine though). But i dont think this would make a difference. Try running a large portion your input DNA out on a 1% agarose gel. If you can't see anything; then /agree there's no DNA there... but you should 1) see DNA and 2) get an idea as to how well your sonications are working out.

Also, i'm relatively new to ChIP myself.. but i work in a lab where people are doing ChIP all day long. First off... on my blog you can find links to two different ChIP protocols currently in use (on the right under "My Protocols") in our group. You might want to check them out if only just to compare. Also, i usually sonicate about 1x10^7 cells per ml in my IP Buffer, but when I check the OD260/280 there is barely anything there at the end of my protocol (maybe 20-30ng/ul of DNA in my final input DNA). I'm using a Nanodrop to measure DNA... but frankly, i've stopped doing this becaue it just seems a waste of time. Instead i just load relative equal amounts (by final volume) for each of my samples in my final qPCR (same volume of input, IgG control, and antibody specific samples - 5ul into each of my qPCR reactions). I usually end up with Ct values for input around 24 - 25, which is good enough.

Keep us posted; i'm curious to hear how things go for you.

A fellow yeastie! Before i read your post I was going to say "did you break up the cell walls" lol.. your protocol looks mostly right by me - although we do 12 cycles for our sonications (different machine though). But i dont think this would make a difference. Try running a large portion your input DNA out on a 1% agarose gel. If you can't see anything; then /agree there's no DNA there... but you should 1) see DNA and 2) get an idea as to how well your sonications are working out.

Also, i'm relatively new to ChIP myself.. but i work in a lab where people are doing ChIP all day long. First off... on my blog you can find links to two different ChIP protocols currently in use (on the right under "My Protocols") in our group. You might want to check them out if only just to compare. Also, i usually sonicate about 1x10^7 cells per ml in my IP Buffer, but when I check the OD260/280 there is barely anything there at the end of my protocol (maybe 20-30ng/ul of DNA in my final input DNA). I'm using a Nanodrop to measure DNA... but frankly, i've stopped doing this becaue it just seems a waste of time. Instead i just load relative equal amounts (by final volume) for each of my samples in my final qPCR (same volume of input, IgG control, and antibody specific samples - 5ul into each of my qPCR reactions). I usually end up with Ct values for input around 24 - 25, which is good enough.

Keep us posted; i'm curious to hear how things go for you.