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Old 09-10-2012, 08:19 AM   #1
Ace5858
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Default Strange Bioanalyzer Results, PLEASE HELP!!!

So I was doing a truseq DNA prep for a bunch of samples. I did PCR on the libraries and then did a gel extraction. The gel was a 2% agarose gel which I ran at 50 volts for about an hour and a half. I then did extractions of 180bp, 350bp and 600bp. I used a sigma gel extraction kit to purify the DNA and then ran the samples on a high sensitivity chip. I qubitted and got between 1.5 and 2ng/ul for all the samples. I got this very bizarre read for nearly all my samples. It's this fuzzy, ladder-like read. So I figured that there was some sort of contamination in my sample. I did a bead purification and ran a second HS bioanalyzer chip and got the strangest bands. In theory, it should have been clean bands, but I got anything but clean bands. I've included my initial gel extraction. It's the best gel extraction I've done. And then the pre-bead bioA run and the post-bead bioA run.

If anyone knows why I have these results, I'd really appreciate your help. Thanks!
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Old 09-10-2012, 09:16 AM   #2
GW_OK
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Are these in any sort of high-ish concentration buffer?
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Old 09-10-2012, 09:20 AM   #3
Ace5858
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You know, after doing the gel extraction, I eluted in EB at 50ul. But I needed to get the concentration down to 20ul to get a good enough concentration of DNA so I used a concentrator centrifuge to bring down the volume to about 20ul. Maybe the salts in the EB got concentrated? That might explain the first bioanalyzer results. But then I followed by a bead purification and got the craziest bands. So I'm not sure what's going on. This was with a brand new hs bioa chip/reagents.
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Old 09-11-2012, 09:55 AM   #4
pmiguel
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You have several problems, I think. Something in your samples, most likely -- dust or ampure beads maybe. Your ladders don't look too bad, although there is some unevenness in the band intensity. So your gel mix may be fine.

Some of your samples are way overloaded and/or have some high molecular weight stuff in them that is carrying over into subsequent lanes.

Also looks like you may have sample issues. Not sure. The evenly spaced spikes I usually attribute to adapter concatamers. But if these are TruSeq libraries, they may just be your spike in controls (if you used them.)

May be other issues occluded by these as well.

Like:
Oh, not sure if the loading dye in your sizing gel, the pH indicator or glass fines from your sigma gel extraction kit might have caused some issues. Ampure should have taken out the dyes, I guess. You might want do a hard spin (like >5 minutes at >14k RPMs in a microfuge) then carefully pull sample from the top of the liquid before doing another bioanalyzer run.

Also, I don't like methods for extracting samples that require heat to "solubilize" the agarose -- sigma page looks like it calls for 60 oC. If you went a little too high on that you might have melted all your dsDNA into ssDNA. It would then reanneal, but mostly at adapter ends only with ssDNA middles. Which might give you somewhat goofy agilent results. (Qiagen I know, can be modified to melt agarose at room temp by adding addition chaotrope buffer. Probably would work for that sigma kit as well.)

(Note, as of 9/25/2012 I updated the link below to link to the new version provided by Agilent. PSM)
Agilent has a pretty good troubleshooting manual they keep here. Worth a look.

Good luck,
Let us know how it turns out.

--
Phillip

Last edited by pmiguel; 09-25-2012 at 04:27 AM. Reason: Updated link to new Agilent M&T guide.
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