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Old 05-09-2018, 01:30 PM   #1
robins91
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Question NGS DNA Library Purification via Dynabeads

Hello, I could use some help from the community.

I have built 48 RAD-Seq libraries using a protocol from Tin et al. (2014) designed for use with museum specimens. I have confirmed that the libraries have been built correctly via vector cloning of a positive control. After amplification using Phusion HF DNA Polymerase (40 cycles), all samples have been run on a gel to confirm that bands are produced around ~200 bp.

Then, I purified the PCR products via Dynabeads MyOne Carboxylic Aid as recommended by Tin et al. (2014). The protocol is as follows:

1. 10 l per sample are removed from the bottle. The storage buffer is removed using a magnetic stand. Then, the beads are washed twice in EB and resuspended in EB maintaining the same volume.
2. 10 l beads, 40 l PCR product, and 100 l of 14.5% PEG-6000 (0.9 M NaCl, 10 mM Tris, pH 6) are added.
3. The solution is incubation at room temperature for 10 minutes. The supernatant is removed using a magnetic stand and discarded. The beads are washed twice with 70% ethanol and air dried at room temperature.
4. Beads are eluted in 15 l EB and incubated for 5 minutes at room temperature. The supernatant is kept.

To quantify, I ran a SYBER I Green Assay on all samples and got values between 0.2 ng/l to 2 ng/l. Next, I ran some samples of varying quantity (but within the range of the chips capabilities) on a Bioanalyzer High Sensitivity Chip. Typically, I rarely get a distribution, and if I do, the concentration is very small. Other times, I am getting unusual distributions that do not seem to have a pattern.

I have tried reamplifying for an additional 20 cycles and purifying again with no luck. I even replaced Phusion with GoTaq, which gave me unusual distributions. I am not sure what to make of this and believe something is wrong with the purification step. Does anyone have any experience with these beads or know of a solution?

I have contacted the manufacturers of the beads (Life Technologies), and they suggest high volume of beads and longer incubation times. I have already tried the longer incubation times with a positive control without any luck.

Thank you in advance.
Attached Files
File Type: pdf Tin et al. 2014.PDF (788.9 KB, 12 views)
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Old 05-13-2018, 07:06 PM   #2
austinso
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Frankly, I think there is a typo in the protocol. It shouldn't be pH 6...it really should be pH 8.

I'm not sure how you were even able to pH Tris to 6.0, since its pKa is 8.3...? i.e. it is no longer a buffer at that pH.
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Old 05-16-2018, 10:24 AM   #3
cement_head
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What would be the disadvantage of using a spin-column purification, or alternate bead based purification? (i.e. AMPureXP or ProNex)?
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Old 05-16-2018, 02:44 PM   #4
cmbetts
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That protocol looks like a scaled down version of the various homebrew AmpureXP recipes out there. It's probably a cost savings measure to avoid the premium on the real deal. I'd agree with the spin column or commercial beads recommendation. You otherwise may end up burning way more cash in troubleshooting than you'd save using the more expensive reagent up front. I'm probably too spoiled by corporate labs, but I find homebrew only saves money if you're going to be running enough reactions in the long run that the math heavily favors DIY.
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Old 05-21-2018, 10:38 AM   #5
cement_head
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Quote:
Originally Posted by cmbetts View Post
That protocol looks like a scaled down version of the various homebrew AmpureXP recipes out there. It's probably a cost savings measure to avoid the premium on the real deal. I'd agree with the spin column or commercial beads recommendation. You otherwise may end up burning way more cash in troubleshooting than you'd save using the more expensive reagent up front. I'm probably too spoiled by corporate labs, but I find homebrew only saves money if you're going to be running enough reactions in the long run that the math heavily favors DIY.
Yeah, we came to the same conclusion. We tried the new Promega ProNex beads and they worked very well, and they are very, very fast at separating. Thanks.
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