[Olaf Blue]

So, how close is this to the Broad/Illumina "Double-sided SPRI" method?

[captainentropy]

I think I need to create a new thread called "The Pippin Prep is dead! Long live the Pippin Prep!"

It is dead. In the lab next to mine (who actually owned it) it's fertilizing a back room full of old broken equipment. That's because I taught them this trick for size-selection that I learned and improved on. Note: this size-selection is post-PCR, which I've found to be vastly better for library construction.

1) Selection for fragments <600 bp
a. Raise the PCR volume to 100 μl with TE and add 65 μl SPRI beads (Beads/DNA = 65/100).
b. Vortex and incubate for 3 mins.
c. Collect beads on magnet and transfer the supernatant to a new tube.

2) Selection for fragments >200 bp.
a. Add 35 μl SPRI beads (This will change the ratio beads/DNA = 100/100)
b. Vortex and incubate for 3 mins.
c. Collect on magnet and discard the supernatant.
d. Wash the beads with 500 μl of 70% ethanol.
e. Dry beads for 5 mins @ RT.
f. Elute DNA in 25 μl EB.
1. save 1 μl for Qubit quantitation
g. Analyze on Bioanalyzer 2100.

Also, if you are certain that the fragments that you made your library from were in a range of 200-600bp then the first step (size-selection <600 bp) is not necessary.

Attached are Bioanalyzer traces showing the output of this method. The red trace is the discarded product from Step 2). The green trace is the recovered (retained on the beads) product from Step 1). The blue product is the fully size-selected library that was later sequenced. Notice that there is a lot of product (most, actually) that is < 600 bp. That's why I say that if the DNA used to make the library is in the range of 200-600 then there's no need for Step 1).

[Isequencestuff]

For Illumina LC we currently use Invitrogen E-gels with a modified protocol for Size Selection: After one post ligation Ampure XP purification we elute in 42.5 ul TE Light and recover 40 ul per sample. On an E-gel cassette, only lanes 2-3, and 7-8 are used (Samples are split in half over two wells 20 ul each.) Once you figure out the timing for elution, it's fairly reproducible. We've found almost no adapter contamination utilizing this method. The downfall is that one can only size select two samples per gel, making it only ideal for low throughput applications. We're demo-ing the Pippin Prep and haven't seen a huge difference in sample quality, but there is a significant difference in sample yield (better recovery with the Pippin versus E-Gel). Cost aside, we might get it for high throughput purposes and better library yield.

[MrGuy]

How does the Ampure method compare in terms of size specificity and stringency?

Has anyone figured out a way to conquer the adapter problem using agarose gels for size selection?

Ampure isn't very stringent. I see a lot of small fragments left over that get in the way. For now, I'm using pre-cast gels that have a precast well to suck out your sample. It's not precise in the manner that a human is the time keeper and an eyeball is the migration measurement device, but it is pretty stringent if you get it right. No gel cleanup.

[maierpa]

Is there a more affordable alternative to this (for those of us without 15K)? How does the Ampure method compare in terms of size specificity and stringency?

Has anyone figured out a way to conquer the adapter problem using agarose gels for size selection? Is there a particular set of gel conditions (e.g. 0.8% regular agarose run at 100V in 1xTBE) that is best for agarose size selection?

No matter how many times I try, my end product is WAAAAY too small, ~180bp...

[pmiguel]

Hi Gary,
Don't forget this thread. What we need is something that will allow us to run strand-denatured DNA to remove small primer dimers that anneal to the longer fragment when run on a native gel. The primer/adapter dimers would be 150 nt or less. The desired fragments would be larger. Like 200-500 nt.

--
Phillip

[SageSigh]

Sage does not have direct experience working with ssRNA in that size range, but we think that our existing 3% agarose cassette might work. We are going to put this in the experiment queue and find out. At the moment we are flat out with Blue Pippin (50bp-50kbp) product launch, so it will not make it into the test queue until later this month. As soon as we have some data, I will drop you a line in this forum. I am wondering if any of our existing customers have tried the Pippin with a 3% cassette for this purpose? Would like to try?

[greenhilly]

SageSigh,

We want to capture a broad pool of ssRNAs from 20-100 nt, not discrete bands. We've only used denaturing PAG so far -- non-denaturing PAG may work.

[SageSigh]

"single-stranded RNA species between 20 and 100-120 nt in length. +/-10nt resolution or better"

We can't achieve that resolution with our existing agarose cassettes, but it is likely that we can achieve it with a PAG cassette. Denaturing gel needed? Do you want to capture specific discrete bands? Or can you specify size ranges, such as a tight cut around 25bp, or all RNA from 75bp to 100bp?

[greenhilly]

we are interested in single-stranded RNA species between 20 and 100-120 nt in length. +/-10nt resolution or better would be nice.

[SageSigh]

greenhilly, can you be more specific as to the RNA sizes you need to separate and elute? Resolution needed? - Gary

[bbeitzel]

There was some discussion on this board last year with someone from Sage about making a denaturing gel cassette. They were asking about what kinds of cassettes would be useful, and that came up. Not sure if they are pursuing it or not.

[greenhilly]

Does anyone know whether any of these platforms (or others) offer urea denaturing gels? We are currently size selecting single-stranded small RNAs with urea polyacrylamide gels, and want to start doing a lot more of this -- we're thinking automating size selection might improve reproducibility.

[captainentropy]

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.

I have in the past using the Qiagen PCR cleanup kit, but after reading greigite's last post I don't. I just setup as many PCR reactions as necessary to amplify the total volume of eluate collected from the Pippin. After that I combine the PCRs and purify. It's definitely overkill as I recovered well over 2 ug of amplified library for each sample. Next time I will use this to my advantage and reduce the number of PCR cycles.

[ScottC]

FYI the Caliper guys quoted us at around $24k for the complete unit and starter set of chips & reagents.

... ugh. Over here, via their distributor, we were quoted a 'special introductory price' of $37K for the first ones in each state, (roughly the same amount in US$), and a 'regular' price I think in the high $40K to low $50K range!

Scott.