bead cleanup problem needs fixing

mpaulsen · 09-19-2014, 12:56 PM

I have been using Illumina's TruSeq RNA library kit for a while now with no issues, but messed up on my latest prep. When doing the AmPure bead cleanup after the PCR amplification step, I accidentally added elution buffer rather than the 80% EtOH wash buffer. I realized the error immediately so I incubated the beads, captured them and saved the supernatant. I now have a library that is very dilute, and probably a little dirty. What is the best way to save this library?

atcghelix · 09-19-2014, 01:31 PM

This isn't particularly helpful now, but I've made that mistake so many times (as well as "eluting" with 80% ethanol...) that I now won't do library preps without putting a red piece of tape on ethanol tubes/boats, and a blue piece of tape on water/EB tubes/boats...

If it were me I'd probably dry down in a vacufuge until you're around the volume you would have eluted in, then quantitate. Then if you have enough sample remaining, do a normal cleanup on them again.

But take my opinion with a grain of salt, I haven't done RNA preps.

kerplunk412 · 09-19-2014, 03:21 PM

Assuming your sample is in a volume of 200 ul, you can add 50 ul of Ampure XP and 250 ul of isopropanol and then proceed according to a normal bead cleanup protocol. The isopropanol will cause pretty much everything to precipitate, so in this case the beads are just there to provide a binding surface for the DNA and don't actually do much to help precipitation. Make sure to mix very well, and it might help to incubate for 10 minutes for the binding and maybe even use some agitation, like a tube rotator.

Other options are (a) to perform a 1x or greater bead cleanup or (b) do a normal ethanol or isopropanol precipitation, but the method I suggested is cheaper than (a) and faster and easier than (b).

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09-19-2014, 12:56 PM	#1
mpaulsen Junior Member Location: USA Join Date: Feb 2012 Posts: 2	bead cleanup problem needs fixing I have been using Illumina's TruSeq RNA library kit for a while now with no issues, but messed up on my latest prep. When doing the AmPure bead cleanup after the PCR amplification step, I accidentally added elution buffer rather than the 80% EtOH wash buffer. I realized the error immediately so I incubated the beads, captured them and saved the supernatant. I now have a library that is very dilute, and probably a little dirty. What is the best way to save this library?

09-19-2014, 01:31 PM	#2
atcghelix Member Location: CA Join Date: Jul 2013 Posts: 74	This isn't particularly helpful now, but I've made that mistake so many times (as well as "eluting" with 80% ethanol...) that I now won't do library preps without putting a red piece of tape on ethanol tubes/boats, and a blue piece of tape on water/EB tubes/boats... If it were me I'd probably dry down in a vacufuge until you're around the volume you would have eluted in, then quantitate. Then if you have enough sample remaining, do a normal cleanup on them again. But take my opinion with a grain of salt, I haven't done RNA preps.

09-19-2014, 03:21 PM	#3
kerplunk412 Senior Member Location: Bioo Scientific, Austin, TX, USA Join Date: Jun 2012 Posts: 119	Assuming your sample is in a volume of 200 ul, you can add 50 ul of Ampure XP and 250 ul of isopropanol and then proceed according to a normal bead cleanup protocol. The isopropanol will cause pretty much everything to precipitate, so in this case the beads are just there to provide a binding surface for the DNA and don't actually do much to help precipitation. Make sure to mix very well, and it might help to incubate for 10 minutes for the binding and maybe even use some agitation, like a tube rotator. Other options are (a) to perform a 1x or greater bead cleanup or (b) do a normal ethanol or isopropanol precipitation, but the method I suggested is cheaper than (a) and faster and easier than (b).