strange CHIP input result - (Jun/16/2006 )
I am new to CHIP assay. As everybody else, I optimized the sonicaiton condition and got the smeared DNA around 600bp. Then I decided to see if I can get the PCR products from the input DNA. I separated the sonicated chromatin (the protein concentration in chromatin is 3ug/ul) into 3 tubes. One tube contained 50ul of chromatin, the second tube contained 10ul of chromatin, and the 3rd tube contained 5ul of chromatin. Here is my following procedure:
1. Bring the chromatin to 300ul by adding elution buffer (1% SDS, 0.1M NaHCO3).
2. Add 12ul of 5M NaCl to all samples.
3. Reverse crosslink by incubating o/n at 65C.
4. Add 8ul of 0.5M EDTA, 15ul of 1M Tris pH6.5 and 20ug of proteinase K. Incubate at 45C for 1h.
5. Phenol/chlorofrom extraction to purify DNA from chromatin.
7. PCR using 2ul of purified DNA as template.
However, I found that only the second tube with 10ul of chromatin has the correct PCR bands, whereas other tubes which contain 50ul and 5ul of chromatin respectively did not have PCR products at all. I used the PCR master mix so that all the PCR condition should be the same except the different template. Do anybody know why only a certain concentration of chromatin can give PCR product?
Also, when I do the phenol/chloroform extraction, I found a chuck of white pellets at the bottom after ethanol precipitation, and this chuck can disolve very fast in water. I do not think this was DNA because I only got about 2ug of DNA so that I should not be able to observe such a big chuck in the tube. So...what is this big soluable chuck thing?
I have noticed the same thing. The chuck of white pellets could be SDS. Although you brought the volume all to 300 ul but the starting material (in lysis buffer of different volume) is different, which could be a factor affecting efficiency of DNA recovery.
We use Qiagen PCR purification kit for the DNA purification after reverse crosslinking and proteinase digestion. The results are very consistent. It also save lot of time and effort. I strongly recommend that. You need to order extra PB buffer because the ChIP sample volume is much bigger than that of a PCR reaction.
I tried using Qiagen PCR purification kit, and I got the PCR products:). Here is the DNA purification result:
From Qiaen PCR purification kit, I got DNA concentration 50ul/ml, 260/230=1.94, 260/280=1.88. Then I used 2ul for PCR, there is a clear PCR band.
From the phenol/chloroform extraction, I got DNA concentration 348ul/ml, 260/230=1.5, 260/280=1.64. Then I used 2ul for PCR , there is no PCR band.
It seems that phenol extraction can give higher yield but low purity. It is possible that SDS is left in the pellet and contributed to the big white chuck. However, it is still unclear why PCR did not work after phenol extraction. Because so many people used phenol extraction for their CHIP assay, could any CHIP researcher tell me if the phenol extraction work for you?
My phenol extraction procedure is:
1. Add equal volume of phenol/chloroform/IAA (pH8), votex and centrifuge;
2. Take out the aqueous phase and add 1/10 vol of 3M NaAc(pH5.2) and 2-2.5vol of 100%ethanol, votex and place in -80C for 1h to precipitate;
3.Centrifuge and wash with 70% ethanol. Dry and add 30ul of water.
By the way, the lysis buffer for chromatin prep contains 1%SDS, 10mM EDTA and 50mM TrisHcl, and proteinase Inhibitor. The elution buffer contains 1%SDS and 0.1MNaHCO3.
I think your PCI purification procedure is right. I did the same before switching to kit. The drawbacks of PCI purification is 1) you have to transfer samples into new tubes after reverse crosslinking and proteinase digestion otherwise your sample will come out of the tube during vortex because the cap no longer fit the tube tightly after several hours of heating; 2) you may lose some or all your DNA pellet during ethanol washes. Although kit purification gives lower DNA recovery, but the recovery is consistent cross samples.
To minimize sample loss during ChIP washes, we now use Qiagen small spin column which comes with the Oligotex System. We load ChIP agarose beads to the small spin column, and do all the washes on the column.