Ampure bead clean up

anna_m · 06-17-2013, 06:47 AM

Hello,
How much drying time do you normally allow when using ampure beads XP to clean up your library preps?

I've been using the standard 15 minutes as mentioned in the TruSeq DNA library prep method (or less if the beads appear to be drying quicker). However, I have recently heard of other labs using just 2 minutes drying time as they believe the beads should be a little wet and sticky.

What does everyone think?

Thanks for your help,
Anna.

chevrm · 06-17-2013, 06:51 AM

15 minutes is a bit overkill. You don't want your beads to "crack" when drying, but you don't want them to have residual ethanol either (effects elution). General rule I use is 2-5 minutes, but just give your plate/tube an eyeball and make sure they aren't wet.

anna_m · 06-17-2013, 06:56 AM

Hi,
Thanks for your reply. 2 minutes just doesn't seem enough. Is there not any ethanol carryover this way? I keep an eye on the beads and usually 10-15 minutes is fine so that the beads appear matt and not shiny.

sara_ahmed · 06-17-2013, 07:42 AM

Yes, 2 mins at room temperature is generally not enough. You can put them in the speedvac for a couple of mins to speed up the drying.

chevrm · 06-17-2013, 08:15 AM

I failed to mention that it depends greatly on what type of plate/tube you're using. In a V-bottom plate, it will take a few minutes longer than a U-bottom because of the more-gradual taper. I agree with the speedvac suggestion if you are using a tubebased approach.

snorberg · 06-18-2013, 05:28 PM

I use 15m as well, but just looked over the manufacture's protocol and found this note...

NOTE: A dry time of >5 min at Room Temperature is optional to ensure all traces of Ethanolare removed but take care not to over dry the bead ring (bead ring appears cracked) as this will significantly decrease elution efficiency.After a visual inspection, I resuspend the beads after 10 minutes.

...so maybe 5 minutes would be safer?

JamieHeather · 06-20-2013, 04:38 AM

Sorry snorberg, but that's a slight (but crucial!) misquote:

Quote:

A dry time of ≤5 min at Room Temperature is optional to ensure all traces of Ethanol are removed but take care not to over dry the bead ring (bead ring appears cracked) as this will significantly decrease elution efficiency.

EvelyneLuyten · 06-20-2013, 04:47 AM

Manually we do it for 15minutes, because if there is still ethanol left, the quality of the library is not good.
With our Sciclone, it's 2-5 minutes on 37°C.
I don't think it's bad when it's cracked, but you must see that the beads won't 'fly away' eg. with use under a flow.

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06-17-2013, 06:47 AM	#1
anna_m Member Location: Edinburgh Join Date: Jun 2013 Posts: 17	Ampure bead clean up Hello, How much drying time do you normally allow when using ampure beads XP to clean up your library preps? I've been using the standard 15 minutes as mentioned in the TruSeq DNA library prep method (or less if the beads appear to be drying quicker). However, I have recently heard of other labs using just 2 minutes drying time as they believe the beads should be a little wet and sticky. What does everyone think? Thanks for your help, Anna.

06-17-2013, 06:51 AM	#2
chevrm Member Location: Boston, MA Join Date: Apr 2012 Posts: 14	15 minutes is a bit overkill. You don't want your beads to "crack" when drying, but you don't want them to have residual ethanol either (effects elution). General rule I use is 2-5 minutes, but just give your plate/tube an eyeball and make sure they aren't wet.

06-17-2013, 06:56 AM	#3
anna_m Member Location: Edinburgh Join Date: Jun 2013 Posts: 17	Hi, Thanks for your reply. 2 minutes just doesn't seem enough. Is there not any ethanol carryover this way? I keep an eye on the beads and usually 10-15 minutes is fine so that the beads appear matt and not shiny.

06-17-2013, 07:42 AM	#4
sara_ahmed Member Location: St Louis Join Date: Nov 2011 Posts: 13	Yes, 2 mins at room temperature is generally not enough. You can put them in the speedvac for a couple of mins to speed up the drying.

06-17-2013, 08:15 AM	#5
chevrm Member Location: Boston, MA Join Date: Apr 2012 Posts: 14	I failed to mention that it depends greatly on what type of plate/tube you're using. In a V-bottom plate, it will take a few minutes longer than a U-bottom because of the more-gradual taper. I agree with the speedvac suggestion if you are using a tubebased approach.

06-18-2013, 05:28 PM	#6
snorberg Junior Member Location: San Diego, CA Join Date: Jan 2013 Posts: 5	I use 15m as well, but just looked over the manufacture's protocol and found this note... NOTE: A dry time of >5 min at Room Temperature is optional to ensure all traces of Ethanolare removed but take care not to over dry the bead ring (bead ring appears cracked) as this will significantly decrease elution efficiency.After a visual inspection, I resuspend the beads after 10 minutes. ...so maybe 5 minutes would be safer?

06-20-2013, 04:47 AM	#8
EvelyneLuyten Junior Member Location: Belgium Join Date: Jun 2013 Posts: 5	Manually we do it for 15minutes, because if there is still ethanol left, the quality of the library is not good. With our Sciclone, it's 2-5 minutes on 37°C. I don't think it's bad when it's cracked, but you must see that the beads won't 'fly away' eg. with use under a flow.