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Samples for ChIP-seq library preparation - DNA concentrations measurement issues (Apr/22/2010 )

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Hi !

I have some issues to quantify my samples after immunoprecipitation by PolII and IgG (control).
I have great PCR and real-time PCR amplifications. PolII quandidate regions amplified at 25 Ct before library preparation (no amplification for negative control regions) and about 10 after (and 25 for negative control regions) library preparation.
So everything looks perfect (enrichment of 50 between PolII and IgG negative control for few candidate regions). But the problem is that when I try to quantify my samples, Nanodrop and agilent chip (nano and pico) show a contamination at 230 (even after the library preparation and so after 5 column of purification !!) and no DNA (concentration of 0)... I don't understand how I can have so great amplification in real-time and a concentration of 0. :D
I need a minimum amount of DNA because I want to do a sequence capture before sequencing.
(I used a lot of cells (100/150 millions ) and antibodies (40ug) for each.)

Is somebody could help me please ? :P

Thanks !!

-doudou30-

Hi :)

I had the same problem with my first library preps.

Now, after gel purification I do a second purification with Agencourt AMPRE XP magnetic beads. It's well worth it.
Hope this helps!

Clare

doudou30 on Apr 22 2010, 05:34 PM said:

Hi !

I have some issues to quantify my samples after immunoprecipitation by PolII and IgG (control).
I have great PCR and real-time PCR amplifications. PolII quandidate regions amplified at 25 Ct before library preparation (no amplification for negative control regions) and about 10 after (and 25 for negative control regions) library preparation.
So everything looks perfect (enrichment of 50 between PolII and IgG negative control for few candidate regions). But the problem is that when I try to quantify my samples, Nanodrop and agilent chip (nano and pico) show a contamination at 230 (even after the library preparation and so after 5 column of purification !!) and no DNA (concentration of 0)... I don't understand how I can have so great amplification in real-time and a concentration of 0. :(
I need a minimum amount of DNA because I want to do a sequence capture before sequencing.
(I used a lot of cells (100/150 millions ) and antibodies (40ug) for each.)

Is somebody could help me please ? :)

Thanks !!

-Clare-

Thanks a lot for your answer ! I am trying an ethanol precipitation and if it's not working I'll try your solution !! :blink:

-doudou30-

doudou30 on Apr 23 2010, 04:17 PM said:

Thanks a lot for your answer ! I am trying an ethanol precipitation and if it's not working I'll try your solution !! :blink:


An EtOH precipitation should work but you may lose more DNA that way.
Good luck! Keep us posted :D
And have a great weekend!

Clare

-Clare-

Clare on Apr 23 2010, 07:42 AM said:

doudou30 on Apr 23 2010, 04:17 PM said:

Thanks a lot for your answer ! I am trying an ethanol precipitation and if it's not working I'll try your solution !! :D


An EtOH precipitation should work but you may lose more DNA that way.
Good luck! Keep us posted :)
And have a great weekend!

Clare


Thanks Clare for your help.
EtOH precipitation showed a little improvement but not anough so I tried the beads.
Same thing : seems improved a little but I still have 260/230 ratio at 0.56 (260/280 is better than at the beginning : 1.7 vs 1.4) with Nanodrop. I don't know if the 10ng/ul that Nanodrop gave me is correct (slope is still weird compared to a genomic DNA at the same concentration). And I am affraid that if I redo an agilent chip it will gill me nothing as the previous times. :P

Have you an other idea ? A phenol chlorophorm extraction ? :wacko:

Thanks !

-doudou30-

Hi again :D

Hmm..I am surprised you are getting a crap ratio after using the AMPURE beads. How are you purifying your DNA post-ChIP and after size-selecting your library?
Don't forget that the nanodrop is inaccurate at the low conc. that one usually sends away for sequencing. Have you run your library on a bioanalyser?

Clare



Thanks Clare for your help.
EtOH precipitation showed a little improvement but not anough so I tried the beads.
Same thing : seems improved a little but I still have 260/230 ratio at 0.56 (260/280 is better than at the beginning : 1.7 vs 1.4) with Nanodrop. I don't know if the 10ng/ul that Nanodrop gave me is correct (slope is still weird compared to a genomic DNA at the same concentration). And I am affraid that if I redo an agilent chip it will gill me nothing as the previous times. :D

Have you an other idea ? A phenol chlorophorm extraction ? :)

Thanks !

-Clare-

Hi ! :)
DNA post-ChIP is purified with chelex, after proteinase K and RNase, and after a QIAquick column. After size selecting it is also a QIAquick (note: I had a lot of small fragments at 100/150pb on my gel, even should be at about 250bp (about 200pb after sonication), degradation ?).
For the AMPure beads (I used AMPure and not AMPure XP), I used a lot of beads to be sure to kept small fragments. Do you follow exactly the protocol ?
I'll try the bioanalyser today (but I am not really optimist). :D

Thanks again,
I'll let you know if it works
:D

-doudou30-

Hi - yes - I follow the exact protocol for the AMPURE clean up.

Let us know what your bioanalyser run looks like.

Clare



doudou30 on Apr 26 2010, 02:46 PM said:

Hi ! :)
DNA post-ChIP is purified with chelex, after proteinase K and RNase, and after a QIAquick column. After size selecting it is also a QIAquick (note: I had a lot of small fragments at 100/150pb on my gel, even should be at about 250bp (about 200pb after sonication), degradation ?).
For the AMPure beads (I used AMPure and not AMPure XP), I used a lot of beads to be sure to kept small fragments. Do you follow exactly the protocol ?
I'll try the bioanalyser today (but I am not really optimist). :D

Thanks again,
I'll let you know if it works
:D

-Clare-

doudou30 on Apr 26 2010, 06:46 AM said:

Hi ! :)
DNA post-ChIP is purified with chelex, after proteinase K and RNase, and after a QIAquick column. After size selecting it is also a QIAquick (note: I had a lot of small fragments at 100/150pb on my gel, even should be at about 250bp (about 200pb after sonication), degradation ?).
For the AMPure beads (I used AMPure and not AMPure XP), I used a lot of beads to be sure to kept small fragments. Do you follow exactly the protocol ?
I'll try the bioanalyser today (but I am not really optimist). :D

Thanks again,
I'll let you know if it works
:D


When you say that you chelex purify, post ChIP, do you heat the DNA to 80C or above? If so, this could leave you with mostly singe stranded DNA since your DNA sample is likely so complex that it won't be able to rehybridize. Obviously this isn't related to your A260:230 problem but it will cause problems when you go to add the adapters for sequencing. You'll end up with a much smaller library than you expect since you'll only be sampling those fragments that managed to rehybridize..

-KPDE-

Yes I used 100C :unsure:
Should I use instead a phenol/chloroform extraction ?
I just not sure to understand why size of fragments is smaller ? (and not just less fragments than expected)

But thanks a lot for this information ! It'll help me a lot ! :rolleyes:

-doudou30-
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