Library size selection

[LacquerHead]

Hi everyone,

Want to size select some Nextera libraries and wondering about everyones preferred method? I have tried Blue Pippin but the cassette % size constraints make this difficult. Moving to trying with gel and want to get an idea of what % of what kind of agarose (e.g. MetaPhor) people are using and running conditions? I am trying to select 150-800bp approximately. Thank you for your feedback!

LH

[jdk787]

Hello Lacquerhead,

I have done a lot of gel size selections with 2% agarose gels which worked well. I used low-melt agarose, and don't think the variety matters too much. If you are selecting down to 150bp you will want to make sure that you let your sample migrate long enough to separate fully from any potential dimers that will be ~120bp.

[LacquerHead]

Don't have anything at 120 in these samples, only around 50bp so shouldn't be a problem. Which kit do you use? Ive heard a suggestion to use regular gel extraction kit but with the MinElute columns.

[LacquerHead]

Another question is how do you stain the gel, EtBr or something else like SYBR Gold?

[jdk787]

I work at Bioo Scientific, so I just use our reagents.
http://www.biooscientific.com/Illumi...Kit-Components but any gel any gel selection kit should be fine.

Both Ethidium Bromide and SYBR Gold will work.

[LacquerHead]

How is the efficiency of the kit in terms of ng recovered? In this lot range of 150-800bp? Thanks.

Originally Posted by jdk787

I work at Bioo Scientific, so I just use our reagents.
http://www.biooscientific.com/Illumi...Kit-Components but any gel any gel selection kit should be fine.

Both Ethidium Bromide and SYBR Gold will work.

[LacquerHead]

Does anyone have good protocols for removing fragments larger than 800-1000bp with Ampure XP beads?

[nucacidhunter]

I wonder why you want to size select a Nextera library in 180-800 bp range. If you sequence a library at this size range 80% of reads will be from fragments below 500 bp and hardly any reads from fragments larger than 800 bp.

I do not have a validate protocol but you would need to do a double SPRI. In principal, add 0.48X bead and take the supernatant then add more beads to make the bead ratio 0.8X. I would suggest doing this with sheared DNA if it is available to make sure that your batch of AMPure beads gives desired results.

[jdk787]

Originally Posted by LacquerHead

How is the efficiency of the kit in terms of ng recovered? In this lot range of 150-800bp? Thanks.

The gel selections that I have done have been before amplification, so I don't have a lot of data on recovery, but would expect >60% recovery.

A bead based size selection should have a higher yield than using a gel, and I agree with nucacidhunter in regards to the upper ratio and using fragmented DNA to validate beforehand.

[Markiyan]

Originally Posted by LacquerHead

Does anyone have good protocols for removing fragments larger than 800-1000bp with Ampure XP beads?

Do a large fragment depletion beads bind at 0.6X - 0.45X and take the supernatant forward (add more beads to supernatant, to capture the smaller fragments - like 1.5X - 2X).

QC by bioanalyser, adjust ratios as needed.