Get rid of big fragments
I need to remove fragments > 500bp and already removed everything <100bp (I basically have a smear that starts around 100-150bp). I would like to try to use 0.7x (from bluescript gel) and keep the supernatant.
What would you do next? Clean-up the supernatant with Qiaquick or MinElute or add 1.8x beads and start all over again?
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Originally posted by molly burns View PostI would like to understand more about double SPRI method to remove small fragments or adapters. I read that if I use 1.8 V of Ampure beads, only large DNA fragments will bind to the beads. On the other hand, if I go sequentially, that is use 0.8 V beads, it will only bind small fragments and large fragments will stay in sup. I can then recover the large fragments by adding another 1 V of beads. How is it possible then to remove small fragments by adding 1.8 V of beads? How does then small fragments stays in solu with 1.8 V of beads?
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want to understand science behind short fragment removal by ampure beads
I would like to understand more about double SPRI method to remove small fragments or adapters. I read that if I use 1.8 V of Ampure beads, only large DNA fragments will bind to the beads. On the other hand, if I go sequentially, that is use 0.8 V beads, it will only bind small fragments and large fragments will stay in sup. I can then recover the large fragments by adding another 1 V of beads. How is it possible then to remove small fragments by adding 1.8 V of beads? How does then small fragments stays in solu with 1.8 V of beads?
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Originally posted by koadman View PostDoes anyone know if it's possible to take the eluate from the pippin prep and load directly to an Illumina flowcell? I'm wondering whether it could be used to simultaneously size-select & cleanup amplification-free libraries.
It worked.
But there are a couple of additional issues for a non-amplified library:
(1) The Y-adapters apparently cause library fragments to migrate aberrantly on some, but not all, electrophoretic conditions. Details are in another thread. I think they may run at their correct size on e-gels, but appear larger than they actually are under most commonly used electrophoretic conditions. After enrichment PCR the adapters are no longer Y-ed, so the effect disappears and you see the amplicon's true sizes.
(2) Just to state the obvious: when you work with very limiting amounts of DNA, you have to watch losses to binding against plastic-ware, etc. A typical microfuge tube may be able to bind 1 ng of DNA (complete guess.) So, if you have 200 ng, who cares. But if you have 1 ng total, you might lose the majority of your library if you don't use low-bind plastic-ware.
--
Phillip
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Pippin prep & amplification free libraries?
Does anyone know if it's possible to take the eluate from the pippin prep and load directly to an Illumina flowcell? I'm wondering whether it could be used to simultaneously size-select & cleanup amplification-free libraries.
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Pippin Prep Max Loading
Back to the Pippin prep topic, the company claim the optimal sensitivity for separation is 10-15ng DNA band. I'm a little concerned to use Pippin to do second purification after PCR since the amount of PCR product is far above the optimal sensitivity, though below the max load limit (4ug DNA band).
I'm wondering if anyone has titrate the separation sensitivity to higher DNA amount 200-4ug band (in 200-500 range). Thanks!
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So artefacts with paired end and TruSeq adaptors seem to make sense, but not those obtained with single end adaptors. What am I missing?
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Hello
I always see adapter dimer at about 123-124 bp. Given that TruSeq index adapters do add up to about 124 (62+62?) with your BioAnalyzer being a mere 1% off, these numbers are correct.
TruSeq protocol has too many cleanup steps and loss of material is inevitable. However, adapter/ PCR mix titration should take care of any imbalance in the final PCR.
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I do single end and the adaptors are 33 and 34bp. If you add primers, it makes 58bp + 34bp + 1 (the A)= 92bp
Does everyone have the same artefact or are they different between single end and paired end?
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Originally posted by odile View PostWould anyone have a diagram with the sequence of the amplified adaptor dimer artefact?
I just don't understand how it can come up to 120-130bp.
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Would anyone have a diagram with the sequence of the amplified adaptor dimer artefact?
I just don't understand how it can come up to 120-130bp.
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Originally posted by jgibbons1 View PostI've had the same issue but routinely use a second gel purification to fix this. Although, you absolutely lose some DNA with the second gel purification. I use the Qiagen Minelute kit to elute with a smaller volume (10-12ul) in order to increase the concentration. This seems to do the trick nicely. I haven't played around with the AMpure beads but will be doing so soon.
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Originally posted by captainentropy View PostHowever, after the elution step there is no need for purification of the DNA prior to the PCR, you will only lose DNA doing that. You can amplify directly from the eluted sample. What I do is I take the elution volume (I elute 200-600bp) and divide it into 36 uL aliquots and how ever many that is is how many PCR reactions I set up for that sample. Usually this is between 7-12. Doing this means you are able to maximize the amount of size-selected DNA in the PCR step resulting in more library. This also means you can get away with fewer amplification cycles thus reducing the number of potential PCR artifacts.
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Originally posted by dagmar View Post@Captainentropy
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Have you ever run a PCR product on the Pippin Prep without a purification step like columns or beads in between?
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If you start out with 100ng you probably are also dependent on the high sensitivity chips. Have you ever had problems running samples from the Pippin Prep on an Agilent? The elution buffer contains much more salt than they recommend for these chips.
2nd question - after the Pippin I use the eluate directly in the PCR with no problems. After that I clean them up using the Qiagen PCR cleanup kit.
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