Picogreen vs bioanalyzer discordance

[vero123]

Hello,

I am wondering if one of you as seen such a problem. I want to do chip-seq and I prepared a chip sample that i concentrated, did a qPCR that gave me a good enrichment (compared with a preimun sample) and did a picogreen to check the amount of DNA i got, before to send to the bioanalyzer to see the size of the fragments that I chipped.

The problem is that the bioanalyzer gave me NO DNA at all (it was a high sensitive kit and done by a core facility that should know how to do it) even though my picogreen gave me 1.5 ng/ul...

The bioanalyzer should have detected it at this concentration. Do you guys have any idea how it can be explained? i'm desperate here... i have been working on that chip for more than a year...

thanks for any input/help!

vero

[huguesparri]

Hello,
Do you have a picture of the Agilent report? If you can include the ladder, it would be great.
Just to know, did you precipitate your chip sample? If yes, what kind of glycogen did you use?

H.

[vero123]

Hi,

I use the qiagen min elute kit to purify DNA after the prot K and RNAse treatment. As you can see in the picture, they do not detect anything and my picogreen was giving me 1.8 ng/ul... I'm thinking now that maybe my DNA is huge and do not enter in the chip for the bioanalyzer. Although my sonication was fine ( when i put my input on agarose gel, i saw a nice distribution of the chromatine where the majority of the fragments where around the 100-300 pb...)

thanks for your help

vero

[huguesparri]

Are you sure the ladder is from the same chip? [FU] for the "start" green peak in the ladder is around 250 and it's only 60 in your sample...

[vero123]

This is what the guy from the core facility sent me. It seems to be coming from the same run of bioanalyzer, since he gave me the picture of the gel too.

When they run the samples, do they mix the ladder, 35 and 10380, in the same lane? That could explain that they loaded less of those ladder with my samples, since I told them I needed the high sensitivity detection.