15 replies to this topic

[doudou30]

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]

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 !!

[doudou30]

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

[Clare]

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 !!  

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

Clare

[doudou30]

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 !!  

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.  

Have you an other idea ? A phenol chlorophorm extraction ?  

Thanks !

[Clare]

Hi again

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

[/quote]

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.  

Have you an other idea ? A phenol chlorophorm extraction ?  

Thanks !
[/quote]

[doudou30]

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).  

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

[Clare]

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).  

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

[KPDE]

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).  

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

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..

[doudou30]

Yes I used 100C  
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 !  

[doudou30]

Ok, it seems that Agilent chip can't read ssDNA, which could explain my problem (good real-timePCR amplification and nothing on agilent)...
I'll try without Chelex and see if it's works...  

[Clare]

I just use the QIAGEN PCR purification columns after ChIP

Clare

doudou30, on Apr 26 2010, 11:51 PM, said:

Ok, it seems that Agilent chip can't read ssDNA, which could explain my problem (good real-timePCR amplification and nothing on agilent)...
I'll try without Chelex and see if it's works...  

[Dukey]

Clare, on Apr 27 2010, 01:22 AM, said:

I just use the QIAGEN PCR purification columns after ChIP

Clare

doudou30, on Apr 26 2010, 11:51 PM, said:

Ok, it seems that Agilent chip can't read ssDNA, which could explain my problem (good real-timePCR amplification and nothing on agilent)...
I'll try without Chelex and see if it's works...  

Yeh i use Qiagen kits too after ChIP, works great and is super fast.

[jamessmith01]

KPDE, on Apr 26 2010, 03:37 PM, said:

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..

Hi KPDE,

Interesting point regarding heating of the samples - would this also apply to other protocols? The Active Motif kit I'm using recommends reverse-crosslinking at 95ºC for 15 mins, which would presumably melt all DNA - would this be irreversible and impact on library preps?

I've now switched to 65ºC for >2 hours, anyway, but it's an interesting point. I also read that it's important not to heat during gel extractions for the Illumina libraries, as this can lead to GC bias in the resulting library.

[KPDE]

jamessmith01, on May 26 2010, 08:22 AM, said:

KPDE, on Apr 26 2010, 03:37 PM, said:

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..

Hi KPDE,

Interesting point regarding heating of the samples - would this also apply to other protocols? The Active Motif kit I'm using recommends reverse-crosslinking at 95ºC for 15 mins, which would presumably melt all DNA - would this be irreversible and impact on library preps?

I've now switched to 65ºC for >2 hours, anyway, but it's an interesting point. I also read that it's important not to heat during gel extractions for the Illumina libraries, as this can lead to GC bias in the resulting library.

Regardless of the protocol, heating DNA for 15 min at 95C will denature the DNA.  If you have a complex mixture (i.e. genomic DNA) then it is unlikely that the DNA will be able to renature to much of an extent.