BioAnalyzer inverted peaks!!

[chipmonk]

I am running my transcription factor chip seq samples in Bioanalyzer HS and getting weird inverted peaks(pic attached)in input samples!! has anybody come across such 'peaks' before?
These are only seen in input samples whereas the pulldown and controls show no bumps at all.Even the tei meth histone L27 pulldown used as positive control fails to show peaks. where as the PCR verification of targets for controls works fine.
I am using HS DNA kit for BA. Magnify kit for pulldown. The kit elutes the DNA finally in 150 ul. which is too dilute to work with. has anyone else worked with this kit and what is the next step to say doing a NGS chip seq.

Thanks

[NextGenSeq]

We saw this too in a ChIP-Seq sample and I'm not sure what causes it. It may be due to the proteins crosslinked to the DNA?

[chipmonk]

@next gen seq

were you able to rectify this and if yes by what means!. could purification again help or is it a problem with incomplete de-crosslinking??

[pmiguel]

My guess would be a dye or fluor of some sort. The fluor used by the bioanalyzer presumably has some background emission level when not bound to DNA or RNA. The emission increases in the presence of RNA or DNA. In the presence of some other compounds one might see a decrease in emission.

You could check with Agilent tech support. They might have an answer for you.
--
Phillip

[chipmonk]

thanks

I have written to tech support. will post if i get a reply!

[julianaect]

I have written to tech support. will post if i get a reply!

I got inverted peaks too. Did you get reply?

Thanks!

[chipmonk]

not much help

hi julian
That was a one off thing i guess. some tech people told it may be because of the remnant magnetic beads. so if you are using dynabead like reagent keep the tubes in rack while pipetting for the BA sample. others speculated there might be excessive protein as well. i think the former may be close to something that may work. all the best.

[FredTam1]

No way, that is a common occurrence for sure. And it's random, which leads me to think it has something to do with an error in the voltage - a machine error. If Agilent won't cop to it, or they keep requesting 'the data' even after a blatant overt description of the graph then it's a common problem inherent to the machine imo

[Olivia16]

We had similar issues in some of our bioanalyzer runs and after working with tech support for about a month they said that it was due to either: 1- dirty electrode pins 2- mistake when making the gel dye mix 3- need a new gasket in the chip priming station....any how we did all the things that Agilent had suggested and the problem got better after cleaning the electrode pins very well and getting a new kit. Therefore it is hard to say if it was the pins or the kit that inverted peaks were coming from. Hopefully that is a bit helpful?

Did you look at the bioanalyzer troubleshooting manual (link below)?
http://www.chem.agilent.com/Library/...MandTguide.pdf

[chipmonk]

not machine issue

thanks for the replies, but when i run these samples that give inverted peaks I also run samples which i know have worked well so random chances or voltage issue or any other machine based issues are ruled out. I think it is the magnetic remnants of the prot-a mag beads. that may cause. i have pipetted using the magrack while loading on gel. hope someone figures out what exactly is happening. but nevertheless my chipseq works!