Phenol/Chlorophorm DNA precipitation after ChIP - (Dec/04/2007 )
So i've been successfully able to execute ChIP in a secondary cell culture system. Now I'm doing ChIP in primary cells -- I'm trying to optimize shearing, but can't get to see the bands. What's up with that?
Basically when I reverse formaldehyde linking by 65C incubation, I get a lot of precipitate. I then digest with proteinase K, and do the whole Phenol/Chlor/Iso purification. The problem is, I can't get a pellet with this purification step... I'm not sure what I could be doing wrong, I think I'm adding enough chromatin (I've tried varying the amount to no effect). Any suggestions would be welcome!!
-MarkER-
QUOTE (MarkER @ Dec 4 2007, 08:51 AM)
So i've been successfully able to execute ChIP in a secondary cell culture system. Now I'm doing ChIP in primary cells -- I'm trying to optimize shearing, but can't get to see the bands. What's up with that?
Basically when I reverse formaldehyde linking by 65C incubation, I get a lot of precipitate. I then digest with proteinase K, and do the whole Phenol/Chlor/Iso purification. The problem is, I can't get a pellet with this purification step... I'm not sure what I could be doing wrong, I think I'm adding enough chromatin (I've tried varying the amount to no effect). Any suggestions would be welcome!!
Basically when I reverse formaldehyde linking by 65C incubation, I get a lot of precipitate. I then digest with proteinase K, and do the whole Phenol/Chlor/Iso purification. The problem is, I can't get a pellet with this purification step... I'm not sure what I could be doing wrong, I think I'm adding enough chromatin (I've tried varying the amount to no effect). Any suggestions would be welcome!!
Is it possible that you just have fewer cells with the primary culture? If you have a smaller amount of DNA you can just add linear acrylamide or glycogen before precipitating with ethanol and that might improve your yield.
-KPDE-
QUOTE (MarkER @ Dec 4 2007, 04:51 PM)
So i've been successfully able to execute ChIP in a secondary cell culture system. Now I'm doing ChIP in primary cells -- I'm trying to optimize shearing, but can't get to see the bands. What's up with that?
Basically when I reverse formaldehyde linking by 65C incubation, I get a lot of precipitate. I then digest with proteinase K, and do the whole Phenol/Chlor/Iso purification. The problem is, I can't get a pellet with this purification step... I'm not sure what I could be doing wrong, I think I'm adding enough chromatin (I've tried varying the amount to no effect). Any suggestions would be welcome!!
Basically when I reverse formaldehyde linking by 65C incubation, I get a lot of precipitate. I then digest with proteinase K, and do the whole Phenol/Chlor/Iso purification. The problem is, I can't get a pellet with this purification step... I'm not sure what I could be doing wrong, I think I'm adding enough chromatin (I've tried varying the amount to no effect). Any suggestions would be welcome!!
I never see a pellet after precipitation (I use primary cells and do not use glycogen). Also, I have switched to using columns to purify my ChIP products - a lot less stessful than nasty chloroform!
-Clare-
QUOTE (Clare @ Dec 5 2007, 02:48 AM)
QUOTE (MarkER @ Dec 4 2007, 04:51 PM)
So i've been successfully able to execute ChIP in a secondary cell culture system. Now I'm doing ChIP in primary cells -- I'm trying to optimize shearing, but can't get to see the bands. What's up with that?
Basically when I reverse formaldehyde linking by 65C incubation, I get a lot of precipitate. I then digest with proteinase K, and do the whole Phenol/Chlor/Iso purification. The problem is, I can't get a pellet with this purification step... I'm not sure what I could be doing wrong, I think I'm adding enough chromatin (I've tried varying the amount to no effect). Any suggestions would be welcome!!
Basically when I reverse formaldehyde linking by 65C incubation, I get a lot of precipitate. I then digest with proteinase K, and do the whole Phenol/Chlor/Iso purification. The problem is, I can't get a pellet with this purification step... I'm not sure what I could be doing wrong, I think I'm adding enough chromatin (I've tried varying the amount to no effect). Any suggestions would be welcome!!
I never see a pellet after precipitation (I use primary cells and do not use glycogen). Also, I have switched to using columns to purify my ChIP products - a lot less stessful than nasty chloroform!
In addition you can try Fast ChIP (Nature Protocols 1(1): 179-185) to avoid using columns or PCI to isolate the DNA.
-KPDE-
Thanks guys, I got it down.
-MarkER-