Low yield after library prep

jhalpin · 01-30-2015, 01:05 PM

Hello,

I am using the Ion Shear Plus reagents kit to prepare libraries for the PGM. I input 200 ng of DNA (measured by Qubit) but am only getting 0.01-0.1 ng/ul at the end of the process. This is enough to continue to sequencing, but I'm wondering if there's anything I can do to improve the yield slightly.

I am shearing the DNA for 7 minutes, which looks really good on the E-gel. It seems like maybe the amplification step is not working? I use zymo instead of the Ampure beads for cleanup and elute in 100 mM Tris.

Thanks,

Jessica

nucacidhunter · 01-30-2015, 03:48 PM

Quote:

Originally Posted by jhalpin

I use zymo instead of the Ampure beads for cleanup and elute in 100 mM Tris.

100 mM Tris is too concentrated and would inhibit many enzymes. If Ion protocol uses 1.8x bead for clean up you can replace it with column clean up. For this application I would use "Q" columns. If the bead ratio is less than 1.8x, it does a small fragment removal as well and column may not be a good replacement for bead.

jhalpin · 02-02-2015, 09:29 AM

Quote:

Originally Posted by nucacidhunter

100 mM Tris is too concentrated and would inhibit many enzymes. If Ion protocol uses 1.8x bead for clean up you can replace it with column clean up. For this application I would use "Q" columns. If the bead ratio is less than 1.8x, it does a small fragment removal as well and column may not be a good replacement for bead.

Well huh. Looking back at my notes I wrote down 10 mM. Not sure when/how that turned into 100 mM. I guess I know why my libraries are weird now.

I'll check out the Q columns, too. Thanks!

jhalpin · 03-06-2015, 07:18 AM

Allright, fixing the Tris concentration didn't fix my problem. I'm inputting 200 ng and getting 0.02 ng/ul at the end of library prep. It's technically enough to sequence but it's really low.. I'd really like to be getting 10 times that at least, and I used to get that much. Any other suggestions??

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01-30-2015, 01:05 PM	#1
jhalpin Member Location: Atlanta, GA Join Date: Jan 2015 Posts: 26	Low yield after library prep Hello, I am using the Ion Shear Plus reagents kit to prepare libraries for the PGM. I input 200 ng of DNA (measured by Qubit) but am only getting 0.01-0.1 ng/ul at the end of the process. This is enough to continue to sequencing, but I'm wondering if there's anything I can do to improve the yield slightly. I am shearing the DNA for 7 minutes, which looks really good on the E-gel. It seems like maybe the amplification step is not working? I use zymo instead of the Ampure beads for cleanup and elute in 100 mM Tris. Thanks, Jessica

03-06-2015, 07:18 AM	#4
jhalpin Member Location: Atlanta, GA Join Date: Jan 2015 Posts: 26	Allright, fixing the Tris concentration didn't fix my problem. I'm inputting 200 ng and getting 0.02 ng/ul at the end of library prep. It's technically enough to sequence but it's really low.. I'd really like to be getting 10 times that at least, and I used to get that much. Any other suggestions??