Version 3 High-Pass Introduction and Best Practices

Sage Science Introduces High-Pass v3 Cassette Definition and
High-Pass DNA Control Kit for HMW DNA Size Selection
April 20, 2014, Beverly MA, -- Sage Science is pleased to release the High-Pass Version 3 cassette
definition and a new High-Pass DNA Control Kit for the BluePippin size fractionation system.
The BluePippin High-Pass cassette definition allows users to collect all DNA fragments of the input
sample that are larger than a specified low threshold size (the “bpstart” value). BluePippin High-Pass
protocols can dramatically improve read lengths on the PacBio RS and RS II systems, by removing lower
molecular weight library elements that load onto SMRTcell waveguides more efficiently than longer
fragments.
The BluePippin High-Pass v3 cassette definition improves on the current v2 cassette definition in the
following ways:
1. Improved accuracy and reproducibility of bpstart values
2. Better recovery of largest DNA fragments of the samples.
3. Accuracy of bpstart value is less dependent on input sample load.
Sheared Genomic Samples
Selected using High-pass v3 cassette definition.
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Best Practices for maximizing HMW DNA recovery from BluePippin cassettes:
1. Wait at least 45 min after termination of the run before removing eluted DNA (it is OK to leave
samples in instrument for hours, i.e. for an overnight run).
2. An additional 10-30% yield can often be achieved by rinsing the elution well with TE+0.1% Tween 20.
Tween solution is provided in Sage Science cassette kits for HMW collections.
To use the Tween rinse method:

Remove eluted sample from elution well at the end of the run

Add 40l Tween solution (supplied with the cassettes) to the elution well

Wait 1 minute

Remove solution and pool with the original extracted sample, or process separately.
3. When removing elution well contents, mix the solution (gently!) by pipetting up and down several
times before removing.
The BluePippin High-Pass DNA Control Kit
The new High-Pass Control DNA (Product No. CHP-7504) is a reference sample designed to help users
get familiar with the High-Pass cassette definition and test performance of alternative size selection
settings. The reference sample is an unlabeled marker ladder containing fragments ranging from 0.5kb –
36kb (approximately 70 ng/band, total 1.25g per lane). Size fractionated control DNA can be analyzed
on agarose slab gels to determine optimal bpstart settings, as illustrated in the figure below. The Control
Kit can also be used to test settings for other BluePippin cassettes (0.75% agarose cassette using marker
sets S1, U1 and T1).
Example of v3 size selections on
the High-Pass Control Kit Samples
Wait, there’s more!
A new cassette definition for High-Pass size selection from 15kb to 20kb is also provided. The new
cassette definition is named 0.75%DF Marker S1 high-pass 15-20kb. Please note that this definition has
not been validated by PacBio.
978.922.1932
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