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AMPure and not-so-simple

posted Jul 8, 2013, 5:48 PM by Kristi Stephenson   [ updated Jul 8, 2013, 5:50 PM ]
As those in the lab will verify, I have an admitted obsession with the AMPure beads. I love to use them for any application possible. They are just so great, and the % recovery is excellent. And they are a lifesaver when it comes to eliminating adapter dimer from next gen sequencing libraries.

We have a ChIP-seq assay in development where a gel punch is required to eliminate the adapter dimers after pcr amplification, and also narrow in on the size of the library we move forward with into sequencing (our target size is around 200-500 bp). When you are processing just a couple samples, the gel punch and isolation is not such a big deal, though it does add an extra 3 hours at least, depending on number of samples. So I got to thinking, what if we used the "double-cut" concept of the AMPure beads to: First, eliminate the really big stuff (top cut), and Second, eliminate the really small stuff (bottom cut). This way I could transfer my entire ChIP-seq library prep to a plate format and greatly increase the number of samples feasibly processed at one time. I did a little experimenting, and here's a summary of what I found.

My basic AMPure protocol goes as follows:

1) Add beads at the desired ratio to the sample and pipet 10x to mix
2) Incubate 15min at room temp
3) Place on magnet 2-5min, until supernatant is clear
4) Remove supernatant (save if needed)
5) Wash beads 2x with 200-500ul 80% EtOH
6) Remove as much EtOH as possible by pipet, then let bead pellet air dry (up to 15min depending on pellet size)
7) Remove from the magnet, add desired elution solution/volume and pipet 10x to mix
8) Incubate 2min at room temp
9) Return to magnet for 2-5min
10) Remove supernatant - this contains the size-selected DNA

First, here is a gel showing the DNA size fragments found in the supernatant (step 4) versus what is eluted off the beads (step 10), for varying ratios of beads to sample. I used a low molecular weight ladder (100bp-1031bp) as my DNA "sample."


Interesting, I thought. You can clearly see that as the bead:sample ratio decreases, the beads selectively bind larger fragment sizes. A nice step-wise illustration where the DNA size lost in the supernatant is mirrored by the size retained on the beads. Also note the beads selectively eliminate the 100bp band no matter what other sizes are retained. This is key for the adapter-dimer removal in sequencing libraries.

Second, based on the gel above I tested different ratios for top and bottom cut, trying to zero in on a combination that could work to select out my 200-500 bp target region for the ChIP libraries.

...

data to come :)
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