[Amiga]

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.

[PaulineF]

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.

[pmiguel]

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.

--
Phillip

[yaximik]

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?

[Amiga]

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.