Pippin Prep for size selection = win

[captainentropy]

Didn't see much discussion of if so I just want to throw this out there: if you have the means to buy, or access to, a Pippin Prep I highly recommend it. It completely solved the problem I often have of the adapter contamination that results in the 124bp peak in the final library (with the Pippin it's no longer there) plus the recovery is MUCH better than gel extraction/cleanup - therefore there is more DNA to be amplified in the PCR step.

The cassettes are $45 each and have four usable sample lanes ($11.25 per lane), so they aren't cheap. But then again quality science isn't either.

http://www.sagescience.com/

[krobison]

How much for the box?

[NextGenSeq]

A far cheaper way to eliminate the 120bp peak is to reduce the amount of primer. We cut it down by a factor of 10 and it disappeared.

[captainentropy]

@krobinson - My lab was quoted $15,000 for the Pippin Prep. That included the machine, monitor, keybpard, mouse, and a starter pack of reagents and cassettes.

@nextgenseq - do you mean adapters? I've used 1:30 and 1:50 adapters and the 124bp band always remained, though in varying intensities. I did try diluting the primers more, 1:10 and 1:30, but that resulted in no products.

Once I tried the Pippin I stopped trying to find the sweet spot for adapters and primers. Plus the yield is so much higher.

To quote Ferris Bueller: It is so choice. If you have the means, I highly recommend picking one up.

[NextGenSeq]

I should clarify that we use lower primer concentration and Ampure XP and it disappeared. Also, our primers are HPLC purified.

[greigite]

We've just started using the Pippin Prep for Illumina library prep as well and it completely solved our adapter contamination issue. Plus, we found that the PP buffer does not inhibit Phusion PCR so we now do size selection post-ligation and put the PP eluate directly into PCR with excellent results. Like captainentropy we also spent a good deal of time messing around with adapter/primer ratios with no useful results.

[captainentropy]

@greigite. That's a very helpful bit of info. Thank you. So you just scale up the PCR volume with the final Phusion buffer concentration still being 1X? (I assume that's what you mean by the PP buffer salts not interfering).

[greigite]

@greigite. That's a very helpful bit of info. Thank you. So you just scale up the PCR volume with the final Phusion buffer concentration still being 1X? (I assume that's what you mean by the PP buffer salts not interfering).

Yes, you can either scale up the PCR volume to use the full PP eluate amount or just use some of the eluate. Phusion buffer concentration is still 1x.

[ScottC]

Anyone have any opinions about other instruments for doing this kind of thing?

[captainentropy]

ScottC, we also tried the E-gel from Invitrogen but it wasn't very good, in our hands, for size-selection for next-gen sequencing. It does look good for cloning though. I think there are a couple more instruments for size-selection coming around. Boreal Genomics has one called Aurora coming out this year, but it'll probably cost much more than the Pippin (I read $30K somewhere). And then there's the LabChip XT from Caliper. This one looks real nice. Smaller elution volume than the Pippin and faster (~30 min runtime). It looks like their kits are designed for size-selecting in a range of 50 to 500bp. I'd like it to collect larger fragments too but this is probably good enough for most people. No idea on the price. They're taking orders but I'm not sure if they are shipping yet. Anyone know?

[ScottC]

Yeah I've heard about the Caliper and Boreal systems too... they look good, although the Boreal system doesn't do any size selection, does it? I thought it was just a purification system (though still very interesting and useful if you need that kind of thing).

I wonder how long it will be before they can select at larger sizes... they say they kits for 10kb 'coming soon'.

[captainentropy]

It's the Aurora model that does, or so it says here

This week, the company is showcasing a new instrument at the Lab Automation meeting that incorporates improvements it has made over the last year or so. That instrument, called Aurora, "has been designed for ease of use," according to the company's website. It uses disposable gel cartridges, and run times have been shortened to 5 to 10 minutes. Moreover, it can size-select DNA "so you can do clean-up and length selection in one shot," according to Marziali.

but that is from an old article and interview, maybe they scrapped that feature?

[dhinerfeld]

Has anyone used the Pippin for mRNAseq libraries using the Illumina-type protocol. On the surface, it should work just fine, but we have had some struggles with getting the PCR to work well after the Pippin.

[LMcSeq]

Do you purify the product after it comes out of the Pippen Prep? There's ethidium bromide in there...perhaps you just need to run it through a column or add more volume of sample to your PCR.

[ScottC]

Anyone using the Caliper machine yet?