cDNA RL weirdness

[RCJK]

Hi all,

I've prepped a couple of libraries, but am having some issues with one of them and do not really know what's going on with it. Both samples were ds cDNA that I input into the cDNA Rapid Protocol at the point of ds cDNA purification (section 3.2.4). After library prep, when running them on a HS DNA chip, sample 1 looks ok, but sample 3 has an uneven baseline and a broad profile. I reran it on another chip thinking it just ran funny, as happens from time to time w/ the HS DNA chips. This didn't make any difference. I repurified the sample by using the Ampure XP protocol for sample clean up (1.8X beads to DNA) and reran that in case there was something contaminating the sample and making it run weird. That didn't make any difference and the sample had a similar trace. Today I took 27ul of the sample and redid the size selection as per the cDNA RL protocol and then ran that on the bioanalyzer. That didn't seem to help either as the sample still shows a broad range of fragments sizes and an uneven baseline. I even diluted the sample and ran it in case the chip was being overloaded, but that didn't change anything.
I'm not really sure what is causing this or what else can be done to salvage this library. One thought was to try and use the Pippen Prep to do a size selection, but we only have the 2% cassettes for selecting 100-600bp.
I have no more original sample to prep the library again as I had to use all of it as input into the protocol.
Any thoughts or suggestions? Thanks in advance!

Jason

[pmiguel]

Can you denature and aliquot and run it on an RNA pico chip? Could be some inter-molecular hybridization going on. Alternatively, denature and aliquot, hit it with RNAseA, then run it on the high sensitivity chip. Could just be a bunch of residual RNA.

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Phillip

[hmmngs]

It definitely looks to be overloaded. Did you do a large dilution? What about trying it on a DNA 7500?

[pmiguel]

It definitely looks to be overloaded. Did you do a large dilution? What about trying it on a DNA 7500?

Overloaded? The spike in peak is 1/3rd the size of the sample peak. Looks like a reasonable loading to me.

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Phillip

[RCJK]

I thought it could've been overloaded as well, so I ran dilutions and the profile looks the same. I don't think it's concentrated enough to be picked up on a 7500 chip.

I've created a library from a different but similar sample and it has the same profile. I am wondering if something is not right with the size selection (sizing solution perhaps, it's a bit expired but has been working fine), so I prepped a gDNA library from another sample to check the sizing on that. HS DNA chip to be run later to check the sizing of that.