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Old 09-14-2009, 08:11 PM   #1
wakskat
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Exclamation Question about 350bp band showing up after PCR

Hi All,

I'm having a weird 350 bp band showing up in my ChIP-seq Input libraries. I am using the Illumina protocol. After size selecting 175-225 bp and then PCRing, a 350 bp band has shown up on my gel in addition to my 200bp band. Does anyone know where this is coming from?

My theory is that after the ligation of the adapters when I clean up the reaction, I elute from the min-elute column with 55 degree Elution Buffer. This higher temp may been generating a 350 bp single stranded DNA that is migrating along with my 175-225 dsDNA. Does this seem like a plausible explanation? I have also been digesting the gel fragments at room temp to prevent GC bias like has been suggested here on the forum.

Thanks for the help and I am posting pics of the pre and post pcr gels.

Mark
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Old 09-15-2009, 08:12 AM   #2
wakskat
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Default Update

It seems that all of my ChIP-seq library samples have this larger band post-pcr but this sample had a stronger band at 350bp than 200bp. I did read on a protocol from UC Davis that a larger band may indicate over PCRing. Does anyone know why this might be?

Thanks,

Mark
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Old 09-18-2009, 07:14 AM   #3
jorgemon
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Smile

Hi Wakskat.
We may be wrong, but this is what we think...
We have had the same problem in some pcr products too and think that probably it's caused if you do too much cycles in pcr...more than 18.
When you have an initial chip-DNA bad fragmented (with only a few sizes between 150-300bp) you´ll get an elution with poor DNA concentration of specific sizes and excessive DNA from biggest sizes. It´s very important test this analyzing a Input sample with bioanalyzer to show the real size distribution.
So, if we combined less specific DNA concentration (be careful when you quantified your DNAs...if you have degradation or nonspecific fragments you'll have a bad estimation of your real DNA concentration...) and too much cycles in pcr, you'll get this fragment around 350-400bp.
You can dilute the pcr primers 1:1 to have the half concentration in pcr or more in order to prevent the nonspecific fragments (we think the primers can generate nonspecific fragments as that...if you don't use a dilution of adapters, you'll get an nonspecific band around 70-90bp after ligation= recombined adapters complex).
Is possible that it happens the same with pcr primers.
But in the other hand, may be possible as you said..."350 bp single stranded DNA that is migrating along with my 175-225 dsDNA".
In this case, we recommend to you, leave your pre-pcr gel running more time for separate your bands all that can be possible.
If you have a good concentration of DNA after pcr, you can scise a band betwenn 150-300bp and re-purify with minielute (you'll lose some DNA but probably you will not get the nonspecific fragment in your sample for sequencing).
Good luck!!!
Regards.

Last edited by jorgemon; 09-18-2009 at 07:25 AM.
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Old 09-18-2009, 08:26 AM   #4
wakskat
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Thanks Jogemon. I used a 1:40 dilution for the adapters and a 1:1 dilution for the PCR with 18 cycles. I haven't checked my input on the bioanalyzer, just ran an agarose gel and looked like a nice smear between 200 and 500 bp. I talked to the genomics core at UC Davis and they have actually cut out and purified this 350-400 bp fragment and could generate clusters and sequence from it. I am running the PCR reaction on a 2% gel and gel purifying the library again, so I am getting rid of most of the 350-400bp fragments according to the bioanalyzer. I hope putting this up on the forum will help others in the future who run into this problem.
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