Double AMPure size selection in Titanium 3kb span paired end library

[H5N1]

In the Paired End Library manual 3kb span (Oct. 2009 version), step 3.11 final library size selection, there is a bouble AMPure size selection on amplified paired end library. I understand the first ampure removes the larger fragment and the second ampure remove the small fragment in order to generate a sharp peak around 500 bp. But I couldn't understand the ratio it uses in the second ampure.

Based on AMPure beads titration, if the PE cutoff value is 0.7, x=70ul. I add 70ul AMpure beads to 100ul PCR product. At this ratio (0.7x), larger fragments (i.e. >600 bp) will bind to ampure bead. Supernatant contains everything <600 bp will be ampured for a second time to remove smaller fragment (let's say <500 bp). My question is on the second AMPure selection. Continue with my example, the supernatant of the 1st AMPure is 170ul, then I add 100ul EB, the total volume is 270ul. How much AMPure is required? Y=X+20=70+20=90ul. 90ul AMPure to 270ul DNA, the AMPure ratio is 0.33x. At such low ratio, how can DNA around 500bp bind to the AMPure beads? Remember at the titration, the ratio tested are between 0.5x to 1X. Even at 0.5x, 500bp bind is mostly removed.

Hope to get some answers soon. Many thanks!

[H5N1]

I know where I am wrong!

Now, I understand where my calculation is wrong. Actually, the ratio of my second AMPure is at 0.8x, not 0.33x. I forgot that in the 170ul supernatant from the first AMPure step, it already contains a portion of 70ul AMPure buffer. What plays the role in size selection is not AMPure beads, but it's buffer. In the second AMpure step, 90 ul plus the 70ul that aleady in, is 160ul. All the non AMPure portion is 100+100=200ul. So the ratio is at 160/200=0.8X

[nextgen]

How much DNA do you recover after two rounds of size selection?

[H5N1]

Hi, I've only done one batch of eight Titanium 3kb paired end library. The yield after double AMPure selection is around 10 ng(most between 9 to 13ng, one extremely low, only 3ng), which is measured by Quant-iT dsDNA HS assay and is pretty low. The PCR product is aound 300 ng, but it might contain the primer dimer.

I knew my library prep has two potential isssues that result in low yield.
1. circularization adaptor ligation might be less optimal.
2. cre recombinase binding issue.

I also noticed after the 2nd AMPure, increasing the EB incubation time/vortex thoroughly would improve the yield.

Btw, I didn't do single strand selection on the lowest yield library (3ng one) since I worry I won't get enough ss library for quantitation. I used the ds library in emPCR and sequencing. The results is pretty good. Personal opinion, without AB adaptor selection, the double strand library works equally well as single strand library. library with AA or BB adaptors shouldn't interfere with the sequencing. Perhaps that's the reason why AB adaptor selection is omitted in Rapid library prep.

[nextgen]

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