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Hello, I recently got a PippinHT loaned to me for a demo, and I am using it for final library pool size selection before loading my library onto a MiSEQ. I am wondering if I can use the Elute directly from the Pippin and dilute/denature for MiSEQ prep. I am concerned that the TE buffer in the Pippin will interfere with sequencing. I have contemplated using a Qiagen Clean up spin column after elution from the pippin, but I am concerned about yield loss. Let me know if anyone has tried going directly from pippin elute to Miseq. Thanks!
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If I may piggy-back on your thread: I'm also wondering whether it's necessary to perform a clean-up step post-Pippin.
I've performed standard gel-extractions before and sent the product straight to sequencing (Sanger, extraction from 0.15% agarose gel) and it obviated the need for post-PCR ExoSAP clean-up.
Does the same logic hold here? ^ -
Pippin elutate contains 50 mM Tris, 30 mM TAPS, 1-2mM EDTA with pH>8. In my experience some polymerases were able to amplify straight and some failed requiring a buffer change. Clustering may be affected if library concentration is low and it does not get diluted before denaturation step.Originally posted by Carcharodon View PostComment
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What volume is your Pippin eluate in? You shouldn't lose too much with a column-based purification, and if you can reduce your volume from say 40 to 12 microliters the increase in concentration should more than make up for any loss during cleanup. And considering the time and expense of sequencing versus performing another cleanup I think it makes sense to clean the product before sequencing.Last edited by kerplunk412; 08-13-2015, 02:54 PM.Comment
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This all makes sense. Thanks everyone!Comment
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Pippin to Sequencer
We have been using Pippin Prep to size select our Small RNA libraries for about 2 years now. We do not perform any additional clean up following elution and have comparable clustering at sequencing. We were concerned initially as well, but testing showed very little variation. Another user said to reduce the elution volume, but that will decrease your efficiency from our experience. We commonly elute small amounts of material, and found that if we reduced the volume, we would lose even more of the sample.Comment
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Hi Steven
I wonder in your experience how Pippin selected small RNA library compare to gel cut regarding recovery of off target size fragments considering that small RNA fragments cover a narrow range of 10 base around 150 bp.Comment
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Currently we run a size range of 125 - 160 bp and are getting far better identification of small RNAs than when we were using standard gel isolation. I attribute this to getting less large fragmented RNAs taking up sequencing clusters, leaving mostly our target to take up the reads. It also helped us narrow in our input concentration for sequencing since the library size range is so consistent.Comment
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Sorry about the late reply...I have been using a concentrator column on my pippin elute to do a buffer change into EB. I found that using a qiagen spin column resulted in a significant yield loss probably due to pH of the pippin elute. I use an Amicon Ultra 30K 0.5ml column and bring up the pippin elute to 500ul in EB buffer,after the spin I collect the ~30ul of elute. I then quantify, dilute to 2nm, denature and load on the MiSEQ at 5pm. I am going to attempt to load pippin elute directly onto the MiSEQ this friday after reading some of your responses. I will let you all know what happens!
**Update**
After running my library through the BluePippin I quantified the elute and needed to dilute it about 15X in EB to reach 2nm for MiSEQ denature. I followed the general MiSEQ loading protocol and loaded at 5pm. My cluster density was about 850 and quality was comparable to libraries I did a buffer switch on after elution from the pippin. I would say it is safe to load pippin elute onto the MiSEQ as long as you dilute your library. hope this helps someone. Cheers!Comment
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I agree it is safe to run as it is. We carry out size selection using the blue pippin instrument on our TruSeq nano libraries after amplification and the final library is the eluted sample taken from the elution well of the cartridge. The libraries are quantified as normal (fluorometry, trace, qPCR) and then put forward for sequencing where they are denatured and diluted as normal. Glad it worked out for you.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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