[odile]

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?

[captainentropy]

take a look at the image posted by pbluescript earlier in this thread. 1.8 volumes isn't represented but I think the point is clear.

http://seqanswers.com/forums/showpost.php?p=39898&postcount=16

[Chipper]

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?

1.8 V will bind all fragments > 100 bp, start with a lower volume (eg 0.4) to bind large fragments and then add more beads to the sup to capture smaller fragments. Dial in the ratio with a 50 bp ladder or sonicated DNA.

[molly burns]

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?

[pmiguel]

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.

I have a partial answer. We tried a few modifications to the TruSeq protocol, one of which was to do the PCR step on non-size selected library, followed by size selection on the Pippin Prep. Our yield of final library was much lower than what we got following the normal protocol. But it was still enough to sequence.

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

[koadman]

Pippin prep &amp; 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.

[Dodu]

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!

[odile]

So artefacts with paired end and TruSeq adaptors seem to make sense, but not those obtained with single end adaptors. What am I missing?

[genemaster]

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.

[odile]

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?

[Heisman]

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.

The paired end primers used are 58 and 61 bp long, and 58+61 = 119, so I've always thought there should be a dimer at ~120 bp but people typically say 130. I don't know why.

[odile]

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.

[monad]

I'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.

2nd gel purification is the best option here, but you lose lots of library DNA. In this case, you can set additional round of PCR will take care of it. Stick with not more than 5 round of PCR. If your bioanalyer peak is good, you will be fine even with 10-15 cycles of PCR.

[captainentropy]

However, 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.

I learned a new trick today with the Pippin Prep. If you leave the tape covering the elution chamber in place you can elute in a much smaller volume, usually around 20-30 uL. This is regardless of the size range being selected.

[captainentropy]

@Captainentropy

...
Have you ever run a PCR product on the Pippin Prep without a purification step like columns or beads in between?

...
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.

1st question - no. I'm only using the Pippin for size selection prior to PCR.
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.