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Old 10-27-2013, 01:42 AM   #1
microphobe
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Arrow SPRI selection at high temperature or with denaturants for ssDNA?

Hi all,

I've heard that SPRI beads also work for ssDNA, and I'd like to do a SPRI bead size selection of ssDNA > 150 nt (for various reasons I won't go into) but I'm a bit worried about the shorter ssDNAs hitching a ride on the longer fragments by non-specific base-pairing at room temperature. I'm using Ampure XP beads. So my question is, can these beads be used at elevated (stringent) temperatures, e.g. 55 - 65 degrees? Or could I use a denaturing agent like formamide to destabilize base-pairing?

After reading this thread: http://seqanswers.com/forums/showthread.php?t=31787 it seems to have been the consensus that even short ssDNA regions in the ladder can mess with the size selection. Any ideas / experiences to share?
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Old 10-29-2013, 08:14 PM   #2
microphobe
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Well if anyone's interested, I did this experiment to answer my own question:

* Washed 40ul SPRI beads (Ampure XP) and resuspended in 80ul 20% PEG8000, 2.5M NaCl
* Divided into 2 x 20ul aliquots of beads
* Each aliquot of beads was mixed with 10ul of TE containing 1ug 1kb Plus Ladder (invitrogen) at either room temperature or at 60 degrees C (prewarming everything) with shaking for 5 minutes.
* Beads were separated 2 min on a magnetic stand and the supernatant saved. For the 60 degrees sample, separation only took 1 minute (I think the heat reduced the viscosity) and the magnetic stand was pre-warmed at 60C. However, it was probably cooling somewhat during separation.
* Beads from both samples were placed on a room temperature magnetic stand for 2 washes with 70% ethanol. Dried beads at room temperature 3 minutes.
* Eluted in 20ul TE.
* Supernate was microdialysed to remove salt
* Equivalent proportions of fractions (~a 3rd) were loaded on a 1.5% agarose gel with EZvision loading dye / stain.

(note: only loaded ~ 75% of the room temperature eluate compared to other samples because I loaded some onto a separate gel! This might explain why the yield actually appears less for room temp.)


Result: The eluate fractions show good yield after capture at 60 degrees C, very similar to room temp although the 100bp band is less well captured. (I wonder if the size-cutoff is also dependent on temperature?) Supe fraction is difficult to interpret probably because of residual PEG after microdialysis.
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Old 02-03-2015, 10:03 PM   #3
Flemmet
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Hi, I was really pleased to see that you had answered your own question as we wanted to find out if the Ampure beads could be used at a higher temp also. We want to use the Ampure for size selection so I was trialling different ratios of ampure to DNA solution. Basically I mixed 200ng 50 bp ladder in 50ul volume with 0.9x, 1x, 1.1x ampure beads. I bound the DNA at either 55oC or RT, by prewarming the DNA solution and the ampure and heating the magnet block on a heat block. Washes were done at RT for both binding temps. I found that I had virtually no recovery of any ladder bands at 55oC (except the 350bp band that is the most abundant in the ladder preparation), and the expected size selection at each bead ratio (ie selectively retained ladder above 150bp at 1.1 ratio, 200bp+ for 0.9x)
So my experiment is much different result to yours??? Questions: You used Ampure beads and washed them and then resuspended in the PEG/NaCl-can you tell me why you didnt just use the ampure as they were?
Also you said that you washed 40ul beads, resuspended in 80ul PEG solution and then divided into 2x20ul? did you resuspend in 40ul PEG not 80ul.
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Old 02-03-2015, 10:22 PM   #4
microphobe
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Hi, well it was a while ago but my best guess as to why I replaced the PEG is probably general paranoia and wanting to know exactly what is in my reactions. I actually divided the 80ul into 4x 20ul aliquots not 2x as I said above.

The ratio of beads : aqueous DNA solution I used was 2:1 (20ul + 10ul) not ~1:1 as you have used, so this seems like a pretty big departure from my procedure. Perhaps the increased PEG and NaCl in my binding reaction is what allowed it to succeed at high temp.

Good luck!
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ampure, library cleanup, size selection, spri, ssdna

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