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Old 01-28-2011, 05:23 AM   #1
pmiguel
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Default Fragment isolation: PippinPrep vs. LabChip XT

We am looking to streamline fragment isolation procedures in the lab -- avoid manually run gels and their attendant mess and labor requirements.

I am aware of a couple of instruments.

(Prices subject to change, or my misinterpretation, obviously.)

The Caliper LabChipXT ~$24,500 instrument.
Consumables list at $300 for a 5 pack of chips. Each chip can run 3 samples + a ladder or 4 samples. So, roughly $15-$20/sample. Run time is said to be 30 minutes for 4 samples. Size range ~100-500 bp.
http://www.caliperls.com/products/labchip-xt.htm


The Sage Science PippinPrep ~$15,000 instrument.
Consumables list at $45/cassette (4 samples) So, about $11.25/sample. Run time 50-100 minutes--currently two types of cassettes (size ranges) available: 1.5% (300-1500 bp) and 2% (100-600 bp).
Also 30-300 bp and 2-7 kb cassettes are said to be released this quarter.
http://www.sagescience.com/


The PippinPrep is a less expensive instrument, but the shorter run times of the LabChipXT probably compensate for that difference.

Any comments? Especially about other available systems.

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Last edited by pmiguel; 01-28-2011 at 10:38 AM. Reason: Updated PippenPrep size ranges.
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Old 02-02-2011, 05:54 AM   #2
pmiguel
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C'mon lurkers! Don't be shy...

I talked to a Caliper rep yesterday. He said a new chip that can collect out to 700+ bp is nearly ready to ship.

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Old 02-02-2011, 06:09 AM   #3
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Default Caliper XT

Hi Phillip

I was lucky enough to have a demo on the XT just before Christmas. I found it very user friendly and the results I obtained from the size selected library were really encouraging.

I was constructing Rapid Libraries for the 454 and had a problem with a FACs sorted chromosome that gave me some library fragments larger than I would have liked (>2kbp). At the time I only had the 500bp cassette so selected for 500bp +/- 20%. The 454 outputs I got back from this size selected library were amongst the best I'd seen. The most significant difference for me was in the average quality score. Normally we get around 31 for a shotgun library but for the size selected library it was over 33.5. Average read lengths were slightly compromised as I would have liked to have selected for a slightly larger fragment and a number of reads had read through to the 3' adapter. But on the whole I have to say that I was mighty impressed.

The guys who did the demo said that the 1kbp cassette would be available early this year and the plan was to introduce a 4kbp to 20kbp version too- this would be great for paired end library construction although I think the stumbling block at the moment is the amount of DNA you can load.

I also tried a half reaction (half DNA and half reagents) 454 rapid library prep and loaded the whole sample post adapter ligation onto the XT and then quantified the recovered library using the Kappa qPCR kits and got enough library molecules back for over 40x full 454 runs. The cost saving here in terms of time and not having to AMPure purify the library or run a HS DNA chip means that in our hands it will pay for itself within a year.

As you can imagine I am trying everything in my power to persuade my bosses that this machine is an absolute must for the lab!

Clarancer
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Old 02-02-2011, 06:37 AM   #4
pmiguel
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Hi Clarancer,

Yes, the Caliper rep made it sound like the 700 bp chip would go out to 1000 bp -- but maybe with not as tight a distribution possible?

But, for Titanium chemistry, pulling out all the library molecules from 600-800 bp would be just fine.

Have you looked at the PippinPrep at all? (Not sure if it is available outside the US.)

Thanks for your response!

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Old 02-02-2011, 06:59 AM   #5
clarancer
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Default 1kbp Cassette

Hi Phillip

the XT data I saw suggested cv's just over 10% for a 600bp product and just over 16% for a 1kbp product. One of the reasons why I think the quality scores went up on my 500bp size selected library was because the fragments were smaller so more efficiently amplified during the emPCR. I would be concerned that these might drop if I selected too large a library so I am guessing there would be some optimising to be done to find the best size to select for.

As far as I am aware the 1kbp cassette is the same as the 500bp cassette they just run it for longer. Is that what you have been told?

No sign of the Pippin this side of the pond so I can't comment. If that changes then I will let you know!

Clarancer
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Old 02-02-2011, 07:16 AM   #6
pmiguel
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I was told the 500 bp chip would be replaced with the 700 bp chip -- possibly without even a catalog number change.

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Old 02-23-2011, 06:46 AM   #7
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Does anyone have any information one the performance of Pippin vs. Caliper XT? We are currently looking into the two options and are on the fence. I just got off the phone with a Caliper rep and the price has come down from the original posted here (now under $20,000). He also said that if we are using it for size selecting libraries it isn't necessary to continually run the ladder so four samples could be run per chip.
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Old 02-23-2011, 07:11 AM   #8
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We bought the Pippin, but have not run it yet. We just realized, however, that one of the most arduous size selections we do--after 1st strand synthesis on the (Ambion) SOLiD Total RNA-seq kits--may not work at all on the Pippin. The SOLiD kit uses 6% Novex TBE-Urea gels stained with SYBR Gold to visualize. Since the product is single-stranded at this point an present in limiting amounts (probably less than 100 ng) I don't know if it would work on either platform.

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Last edited by pmiguel; 02-23-2011 at 07:29 AM.
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Old 03-01-2011, 10:43 AM   #9
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We just got our Pippin Prep set up a couple of weeks ago. We haven't used it for any library preps yet, but have played around with it to see how efficient it is at isolating different fragment sizes.

Right now it comes with 2% (for 100-600 bp fragments) and 1.5% (300 - 1500 bp fragments) agarose cartridges. Total run time for both cartridges is 2 hours, but the run can be stopped early if your fragments have been eluted. For example, yesterday we tried a 1.5% run to isolate 300-400 bp, 500-700 bp, and 800-1000 bp fragments from nebulized gDNA. The 300-400 bp fragments were eluting around 1 hr into the run, and the 800-1000 bp fragments were eluting at around 1.5 hours, after which we stopped the run and collected the fragments. All samples gave nice smears in the correct size ranges on a gel.

I am hoping that our sequence qualities will go up when we start using this for library preps, as we tend to get a lot of short reads after bead size selection.

First impressions are that the Pippin prep is easy to set up and run, and seems to work as billed. I'll post more after I see how it affects our sequencing runs.

-Brett
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Old 03-03-2011, 10:18 AM   #10
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We are also looking at what is available for sized fragment isolation and are considering these two options. Would the inclusion of ethidium bromide in the Pippen Prep elutes cause problems for any of the Roche 454 library preps? Am I missing that Pippen has gel cassettes labeled with something other than ethidium?

Thanks!

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Old 03-04-2011, 01:05 PM   #11
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We've been using the Pippin for Illumina library preps. The goal is to get a size distribution that's tight enough to allow us to assemble overlapping 100 bp PE reads into a single ~150-175 bp higher quality read. We use the Pippin with 2% cassettes following adapter ligation as that allows us to simultaneously size select the library and get rid of excess adapter. These libraries haven't been sequenced yet so I can't say how well it worked, but the size distribution looked quite nice on a Bioanalyzer and we got almost entirely rid of those pesky ~130 bp amplified adapter dimers in the final product. We cloned the library and Sanger sequenced a few clones to check adapter integrity, and it looked fine. So far I am quite pleased with the Pippin. Huge, huge improvement over gel extractions and I haven't noticed any problem relating to the EtBr.
I haven't used the Caliper XT so can't comment on a direct comparison.

Last edited by greigite; 03-04-2011 at 01:07 PM. Reason: addition
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Old 03-07-2011, 05:16 AM   #12
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We are also considering the options for Illumina library preps as we may soon be involved with those library preps for an off-site HiSeq 2000. But we were very interested in the possibility of using the Pippin for preparing Roche 8 kb paired-end libraries (not to be confused with the Illumina paired-end - equivilant to the Illumina mate pair library preps). The Roche paired-end manual states not to use ethidium bromide in the gel for the gel extraction part; it may be just to avoid the use of UV for illumination and subsequent product damage. I'll send a note to my tech rep and see if I can scare up a response and let you know if I get any info.
Thanks for all the good info.
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Old 03-07-2011, 08:22 AM   #13
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When asked of a key difference in the platforms, Caliper stated that everything your DNA touches on the LabChip XT is disposable, whereas on the Pippin the electrodes are reused.

Can someone confirm this? If so, how do you clean the electrodes between samples? Has anyone looked carefully for carry-over?
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Old 03-07-2011, 09:11 AM   #14
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On the PippinPrep you use a casette, which is basically a fancy agarose gel, with ports to recover the eluate. The casette is disposable (and should be disposed). That's it. It supplied we the appropriate molecular marker and loading buffer. The cleaning step (of the machine, not your sample which doesn't need cleaning after the elution) involves using an empty casette, filling it with ddH2O and leaving it in the machine for 5 min... I have yet to see cross-contamination.

PippinPrep is very easy to use. It's fast. And the yield and purity are excellent. It beats gel extraction with Qiagen or Invitrogen kits. I honestly don't know why not more people are using it. They now have 1.5%, 2% and 3% gels to take of size from 25 bp to 2 kb. I wouldn't pay for the Caliper, it's clumsy compared to PippinPrep. I have nothing against Caliper, I actually think they make very good products, but in this case there's a better technology out there.
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Old 04-14-2011, 12:47 PM   #15
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Quote:
Originally Posted by Amiga View Post
On the PippinPrep you use a casette, which is basically a fancy agarose gel, with ports to recover the eluate. The casette is disposable (and should be disposed). That's it. It supplied we the appropriate molecular marker and loading buffer. The cleaning step (of the machine, not your sample which doesn't need cleaning after the elution) involves using an empty casette, filling it with ddH2O and leaving it in the machine for 5 min... I have yet to see cross-contamination.

PippinPrep is very easy to use. It's fast. And the yield and purity are excellent. It beats gel extraction with Qiagen or Invitrogen kits. I honestly don't know why not more people are using it. They now have 1.5%, 2% and 3% gels to take of size from 25 bp to 2 kb. I wouldn't pay for the Caliper, it's clumsy compared to PippinPrep. I have nothing against Caliper, I actually think they make very good products, but in this case there's a better technology out there.
Dear Amiga,

We are demoing both the Pippin and LabChip XT. However, we find that our yields are better with traditional agarose gel extraction. Can you please provide numeric examples of how much of your programmed size range do you load versus how much you get back in your eluate? With our Pippin demo unit, our best trial was a 30% recovery when only the programmed size peaks were summed up from the Bioanalyzer as input amount. We used the 1.5% cassette, set for 250-300 bp elution with a gDNA sample sheared with Covaris' 300 bp conditions, we were told setting 250 bp (a bit below the 300 limit) was not detrimental, indeed it had been our best yield. It seems others are having much better luck, so maybe we are doing something wrong?

To the general group,
We actually got better yields from Caliper's XT on a first run, already 30% (the 30% off Pippin was on one sample from run #3) and XT uses dry electrodes (no contact with your samples, or any liquid) so there is no cleaning. However, maximum input is 1ug and it has 4 lanes total so if you want to load a lane of their ladder, you can only load 3 samples. I seem to be getting the impression that people generally like these automated fractionating systems but what criteria are you judging by? In terms of yield, we seem to have better luck with agarose gels, but are those of you who prefer these systems judging by the data you get?

With the XT, since maximum input is 1 ug, do you still start your library prep with 5 ug for shearing? Do you dilute your sample before loading onto the XT? We generally do not quantify our libraries right before size selection, so we would also like to know if people do a standard dilution when they load the XT if they started with 5 ug sheared gDNA?
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Old 04-14-2011, 07:12 PM   #16
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Hi Pauline,

The worst I got was actually 30% and this was using a cassette that fail the test the user is supposed to do every time.

Typically I get around 70% (same target size than you or lower). Amount of DNA doesn't seem to matter.
I always change the elution buffer in the casette.

However, I find that when I run a gel after the Covaris shearing for QC purpose the size I input in the PippinPrep are sometime different. I haven't troubleshooted this, but it could be that the different reagents (i.e. different kits to extract gDNA) could impact the precision of the run.

The advantage of not using an automated system is that you see your material and the ladder. You could run the PippinPrep "manually". I would suggest yo and try to extract bands in a DNA ladder to get familiar with the process before extracting your real material this way.

My recommendation is to contact Sage Science directly, they are very nice.
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Old 04-14-2011, 07:47 PM   #17
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Dear Amiga,

We also replace the buffer in the elution wells everytime and we have been trying to troubleshoot with our distributor and Sage Science. We have gotten one response directly from Sage but they just said not to miss the size of the actual sample (we ran PCR products as well) and to use the broad range extraction rather than tight band setting. However, upon trying all their suggestions, the only improvement we got was a more consistent elution volume of around 60 ul. Thank you for your suggestions though! If we manage to keep the unit any longer I would try it.
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Old 04-15-2011, 03:37 AM   #18
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One issue would be that samples loaded in different salt concentrations result in different mobilities on agarose gels. So if salt/buffer concentrations are different between your sample and the Pippinprep ladder...

We too have had cases where the Pippinprep seems to have "missed the mark" -- taking a DNA size fraction different from what was asked of it. But being a little off on the size is probably only going to be an issue if you have a narrow band that you want to get and then miss it altogether because you set the size range too narrow.

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Old 10-26-2012, 04:50 AM   #19
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Hi all,

The last post in this thread is about 1.5 years old and, while the shared information is already very helpful, more experience was certainly accumulated since then. As I am on market for a better size selection approach, I would appreciate more input on the topic. Also, Blue Pippin now offers pulsed-field for large fragment isolation - can anyone comment of this, does it really worth price increase? LabChip XT does not seem to be near offering anything for longer than 1000 bp, correct?
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Old 10-26-2012, 05:52 PM   #20
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The main problem with large fragment selection is that the CV% can be up to 20%. So at 5kb, you have to expect +/- 1kb... Not super precise.
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