PCR after Sonication... - (Oct/04/2005 )
Hi, I'm just trying to set everything up before I do the immunoprecipitation. I did a PCR after Sonication (500bp average) and tried to amplify a 244bp fragment. However I was unsucessful on two occasions. I also included the non sonciation control (genomic DNA not sonicated) but no fragment either. Finally, I used a plasmid with the sequence, and got the band so its not the general PCR conditions.
So what I did - Crosslink, sonicate, reverse crosslink, (no proteinase K treatment), then phenochloroform DNA precipitation, and RNase treatment, then PCR for the fragment. Any suggestions why I'm not getting the fragment in the sonication and no-sonciation control?
Im assuming its the 4hour reverse crosslinking...im going to deterimine if this is the case...thanks.
That sounds weird. Your procedure looks fine. Yes, check if the temperature was right for the crosslinking. If DNA had not been lost during purification, the problem might lie in your primers. I usually have at least two pairs of primers for each locus. Because ChIP usually focus on promoter regions which is generally GC-rich. Some primers may not work.
I have lots of genomic and sonicated DNA (0.5ug/ul). But maybe some residual proteins are hanging around that are inhibiting Taq. The primers should work, they work when using a plasmid with the target sequence.
I didnt spin the cells down after incubating for 4 hours at 65 degrees, and I didnt digest with protinase K. I went right to phenol chloroform/ethanol precipitation. Right now the samples look like any other sonicated ChIP gel but PCR dosent work. I dont think the reverse crosslinking is playing to much of a role sine the average size of the DNA is around 500nt.
Do you think by not spining down the cells and not digesting with protinase K that I am inhibiting the PCR reaction? I have the chromatin suspended in TE buffer with some RNase in there...and I use 2ul of this for a 25ul reaction. Just puzzles me that the PCR isnt working.
Thanks for the thoughts.
the other thing could be phenol contamination.
if you are able to see the DNA on the gel you should be able to spec it, and this will give you an indication on how pure your sample is through the A260/A280 ratio.
After fixation step the DNA was crosslinked with lots of stuff at proximity. I think you have to reverse the crosslink and perform protease K digestion to revover DNA, thereby polymerase can work properly. Although the PCR denaturation step got high temperature (e.g. 95 C) but it's not guaranteed all crosslink could be removed.
Thanks for all the replys...Im still having problems getting the PCR to work with the imput control.
I sonicate (ran this after RNase treatment) and get a good smear less than 1kb. Then I spin down, and dilute according to the upstate protocol. Using the supernatant as the template for pcr, 1, 2, 5, 15, 25 ul, I dont get the PCR product.
Also, it seems after I treate with protinease K and then phenochlorophorm I lose my dna?? Do i have to add Na acetate b4 the phenol step. I never usually do and it works? This stuff is kinda stumpping me...any thoughts would be appreciated...
you add NaAc after your phenol step. Also because you are working with a potentially small amount of DNA, I would suggest you add 20ug of glycogen to aid in precipitation. Glycogen is inert and will not afftect downstream applications such as PCR.
Thanks for the suggestions nick...
I used 500ul (they recommend 20ul) of the 2000ul dilution (I froze the dilution fraction) upstate recommends to reverse crosslink with NaCl (over night) then I treat with 100ug/mL of prot K for 1 hour at 45 degrees (in the dilution buffer) and the phenol chloro ethanol precip.
So, I think I sould have alot of nucleic acid (Mixture of DNA and RNA) making a pellet. But I dont see anything. I ran 1ug (according to A260) and dont see anything on the gel (might be expected since the 1ug is fragmented).
Any thoughts on this...I will run the pcr tonight with 1ug of this material, 1ug according to the UV spec.
By the way, no clean up of the diluted fraction results in no pcr fragment...Im lost...lol
I think i solved this problem. After my formaldehyde treatment the cells are not lysing with the lysis buffer alone. Must be my formaldehyde treatment, if anyone has some problems with their begining steps of the CHIP just ask me...lol...
how did you work this out? When we lyse our fixed cells we just look for the cloudiness of the suspension from the cells to dissappear which means they have lysed.