Samples for ChIP-seq library preparation - DNA concentrations measurement issues (Apr/22/2010 )
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... 
-doudou30-
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...
I'll try without Chelex and see if it's works...
-Clare-
Clare on Apr 27 2010, 01:22 AM said:
I just use the QIAGEN PCR purification columns after ChIP
Clare
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...
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.
-Dukey-
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.
-jamessmith01-
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.
-KPDE-
my problem was effectivelly the fact that I used high temperature. I remove this step and it works very well now (and I avoid to heat above 70ºC)...
-doudou30-