Ampure XP size selection

[dwarner]

I'm wanting to do a size select with Ampure XP beads for around 330 bp. I've got one that centers there, but the peak is quite wide. Does anyone happen to have this worked out?

Thanks.

[pbluescript]

Ampure size selection peaks are generally wide. You can get tighter selection with smaller ratio differences of the beads, but you'll hit a limit in your ability to pipette accurately. If you want tighter peaks, you'll need to use agarose electrophoresis or ideally a Pippin Prep or Caliper XT.

[dwarner]

I already do Ampure selection for 200 bp with the lower selection around 170bp and the upper at around 220. That's tight enough for what I want. What I've got at 330 bp currently is more like 200bp-500bp. I'd like to tighten that up and was just hoping that someone had already done the work to validate that. If not, I guess I know what I'll be doing.

Thanks.

[mbekram]

I already do Ampure selection for 200 bp with the lower selection around 170bp and the upper at around 220. That's tight enough for what I want. What I've got at 330 bp currently is more like 200bp-500bp. I'd like to tighten that up and was just hoping that someone had already done the work to validate that. If not, I guess I know what I'll be doing.

Thanks.

I'm trying to get a selection in the range of 200-250bp, which is close to the 200bp range you mentioned about. Could you please let us know the ratios you used? May be I can try with our lot of Ampure beads and tweak a little bit to get the range I'm looking for.

Thanks.

[dwarner]

1 volume Ampure XP beads.
transfer the supernatent to a new tube and add 0.6 volume beads
discard the supernatent
wash with ETOH (70%)
Dry and then resuspend in water or TE

[TimLar]

Hi all,
I've been doing some library prep optimization recently and titrated PEG (or Ampure XP beads) to DNA sample ratio. This is similar to previously reported studies, particularly Lundin et al 2010, but I used slightly different buffers and wanted to get all in one place, hopefully you'll find it useful. The results are below. I was mostly looking for the conditions that would separate adapter/primer dimers from the main library (i.e. 200bp+ products) in order to implement 'with-beads' library prep approach introduced by Fisher et al, Genome Biology 2011.

Experiment setup:
1) 20 mcL of 0.5 mg/mL Fermentas GeneRuler LR ladder (25bp-700bp) were diluted in 200 mcL TE/0.05% Tween.
2) Ampure XP beads were isolated from 200 mcL of the original suspension and washed twice with TE/0.05% Tween.
3) beads (2) were resuspended in 220 mcL of mixture (1) and partitioned into 10 aliquots, 20 mcL each.
4) to each aliquot a variable amount of 20%PEG8000/2.5M NaCl was added - the volume was titrated between 12 mcL (0.6x) and 48 mcL (2.4x).
5) the binding was allowed to proceed for 5 min at +30C with shaking (1400 rpm).
6) Beads were collected on a magnet, washed twice with 80% EtOH, air-dried (5-10 min) and resuspened in 30 mcL of TE/0.05% Tween.
7) 1 mcL of each eluate was run on Bioanalyzer DNA-1000 along with 1 mcL of input, i.e. mixture (1). Size selection is shown below.

[TimLar]

Titration of Ampure XP beads to aqueous phase ratio.

Experiment setup:
1) 10 mcL of 0.5 mg/mL Fermentas GeneRuler LR ladder (25bp-700bp) were diluted in 100 mcL TE/0.05% Tween.
2) Mixture (1) was partitioned into 5 aliquots, 20 mcL each.
3) Variable amount of Ampure XP beads suspension was added to each aliquot - the volume was titrated between 18 mcL (0.9x) and 28 mcL (1.4x).
4) the binding was allowed to proceed for 5 min with shaking (1400 rpm).
5) Beads were collected on a magnet, washed twice with 80% EtOH, air-dried (5-10 min) and resuspened in 30 mcL of TE/0.05% Tween.
6) 1 mcL of each eluate was run on Bioanalyzer DNA-1000. Size selection is shown below.

[TimLar]

Ligation mixtures of most library prep kits do contain some (often unknown) amount of PEG to enhance ligation efficiency. Therefore, after adding PEG/NaCl as part of Ampure beads suspension or separately (if using alternative beads) it is difficult to predict the final PEG concentration. To address this issue, I titrated PEG to ligation mixture ratio using components of illumina TruSeq kit.

1) To mimic standard RNA-seq library prep procedure 10 mcL of 0.5 mg/mL Fermentas GeneRuler LR ladder (25bp-700bp) were combined with the following components:

• 25 mcL A-tailing master mix
• 10 mcL Ligation STOP buffer
• 35 mcL TE/0.05% Tween

mix well

• 5 mcL of Ligation master mix

total volume is 85 mcL

2) Ampure XP beads were isolated from 60 mcL of the original suspension and washed twice with TE/0.05% Tween.
3) beads (2) were resuspended in 85 mcL of mixture (1) and partitioned into 6 aliquots, 10 mcL each.
4) The following components were added to individual aliquots:

• nothing
• 2 mcL of 5M NaCl (0.8M final)
• 5 mcL of 20% PEG8000/2.5M NaCl (0.5x ratio)
• 7 mcL of 20% PEG8000/2.5M NaCl (0.7x ratio)
• 9 mcL of 20% PEG8000/2.5M NaCl (0.9x ratio)

5) the binding was allowed to proceed for 5 min with shaking (1400 rpm).
6) Beads were collected on a magnet, washed twice with 80% EtOH, air-dried (5-10 min) and resuspened in 15 mcL of TE/0.05% Tween.
7) 2 mcL of each eluate was run on Bioanalyzer DNA-1000. Size selection is shown below.

As such, separation of adapter dimers (ca 100bp) from ligation mixture should be carried out at 0.5x-0.6x PEG ratio. This should, at least in theory, allow to use a single purification step instead of two sequential purification steps employed currently in most protocols. Note, I have not tried it in practice yet.

[zhangyuanyuan]

useful information, Thanks a lot.