MinElute columns for TruSeq preps?

[bbeitzel]

Is there any reason why you can't use MinElute columns instead of Ampure beads during a TruSeq DNA library prep? I can't see any reason for using beads instead of columns after the end repair and A tailing reactions. I can see maybe using beads after adapter ligation to remove adapter dimers, but couldn't you accomplish the same thing by using a MinElute purification followed by gel purification to remove dimers? I know that if you have a lot of adapter dimers that some of those may contaminate larger gel fractions, but is that contamination much of a problem during the limited cycle enrichment PCR?

I am new to TruSeq library preps, so I am trying to understand the reliance on Ampure beads when MinElute purifications are so much faster (compared to the bead steps in the TruSeq protocol.)

Thanks for any thoughts/info.

[protist]

Re MinElute columns for TruSeq preps?

There should not be any reason not to. Using the beads for each reaction allows automation thus a more high-throughput libaray generation process.
If you look at the TruSeq DNA Sample Prep kit info you will see for gDNA libraries they still recommend gel isolation followed by a MinElute clean-up - for the TruSeq RNA libraries all clean-ups or size selections are bead based.

[lterhune]

On a related note-- does anyone know why there is a double purification with AMPure beads after the ligation? This seems unnecessary at first glance, especially when you are gel purifying at the next step.

[protist]

On a related note-- does anyone know why there is a double purification with AMPure beads after the ligation? This seems unnecessary at first glance, especially when you are gel purifying at the next step.

For the TruSeq RNA Sample Prep Kits you don't do gel isolations, all clean-up steps are bead based.

[lterhune]

@protist: Sorry for not clarifying-- I was talking about the TruSeq DNA sample prep kit. We always do the gel option, not gel-free.

[greenhilly]

Is there any reason why you can't use MinElute columns instead of Ampure beads during a TruSeq DNA library prep? I can't see any reason for using beads instead of columns after the end repair and A tailing reactions. I can see maybe using beads after adapter ligation to remove adapter dimers, but couldn't you accomplish the same thing by using a MinElute purification followed by gel purification to remove dimers? I know that if you have a lot of adapter dimers that some of those may contaminate larger gel fractions, but is that contamination much of a problem during the limited cycle enrichment PCR?

I am new to TruSeq library preps, so I am trying to understand the reliance on Ampure beads when MinElute purifications are so much faster (compared to the bead steps in the TruSeq protocol.)

Thanks for any thoughts/info.

Had the same question. If you try the minElute in lieu of AmpPure beads, please let us know how it goes.

[protist]

@protist: Sorry for not clarifying-- I was talking about the TruSeq DNA sample prep kit. We always do the gel option, not gel-free.

My bad assuming it was RNAseq. I still do the gel selection for all gDNA libraries and MinElute prior to the gel isolation step. There should not be a problem and I agree with the gel option there should be no requirement for a double bead selection.

[Veleno]

Less loss of material.

[riehle]

Hello...
I came across the same question that you were posting last year: Can the Ampure Step in the Illumina DNA Sample prep protocol be replaced by MinElute Columns?
Have you tried this out by now? Would you use for elution of the DNA from the columns the volumes indicated in the Illumina protocol or is there some adjustment needed?

[RNAseqer]

You can't efficiently remove the longer style adapters with columns. That's why beads are required.

[pmiguel]

Also if you have enough samples to process in a 96-well plate with a multi-channel pippetter and a good plate magnet, then AMPure is, by far, less time consuming than working with individual tubes.

--
Phillip