Pippin Prep : automated DNA size selection and collection

Product Description

The Pippin Prep is a preparative electrophoresis platform that separates and extracts DNA fragments. Using pre-cast and disposable gel cassettes, DNA is automatically collected in buffer according to software-input size ranges. DNA fractions (typically adapter-ligate NGS libraries) are removed using standard pipettes. Workflow requires about 1-2 minutes of hands on time per sample, and cassettes are available for extractions between 50bp and 8kb. Typical run times (four samples per run) are between 50 and 100 minutes.

Improved library construction for Next-Generation Sequencing

• Significant amounts of time and labor are saved compared with manual prep gels
• Extraction of narrow size ranges streamline sequencing and data analysis
• Accurate size ranges are selected with software, and reproducibly extracted from samples
• High sample recovery reduce amplification cycles and provide more unique molecules
• Minimal low molecular weight contamination improves adapter clean-up

Size selection ranges

Several gel types are currently available. Cassettes are pre-cast and disposable. DNA is optically detected with ethidium bromide. Ethidium bromide-free gel cassettes are also available with pre-labeled DNA markers.

Agarose percent %	For ranges between
3.0	50 - 200bp
2.0	0.1 - 0.6kb
1.5	0.3 - 1.5kb
0.75	2.0 - 8.0kb

 

How the gel cassettes work

Gel Cassettes use an external DNA size standard run in a dedicated sample lane to determine the timing of size-selected collections. Four lanes are used for samples, and one extraction can be collected per per lane according to user input size ranges. Samples lanes are physically separate and tapered to shorten run times and improve resolution.

Gel cassettes

Gel cassettes are pre-cast with agarose gel and are disposable. Each cassette has 4 sample lanes and 1 lane on which an external standared (DNA size markers) must be run. Each cassette order includes sufficient DNA marker for every cassette, loading solution, and spare electorophoresis buffer. A P200 pipettor and tips are also required.

Sample lanes are separated by a physical barrier, and one cut can be automatically extracted per sample. In a less automated fashion, consecutive cuts may collected.

Performance

For smaller fragment targets, extracted size distributions may be as low as 3% if expressed as CV. Below is an bioanalyzer result of a 200bp cut from a 2% gel cassette.  Broader size ranges may also be collected.

Time to collect DNA from samples run the Pippin Prep take between 45 minutes and 2 hours, depending on the sizes range collected and type of cassette used. Hands-on time is 1-2 minutes per sample. Below, the table show relative guidelines - times may vary up to ±5 minutes.

Sample recoverises range between 80-90%, for smaller fractions and 50% for larger fractions on a given cassette.

• 3.5% CV at 100bp
• 4.0% CV at 600bp

1.5% Gel Cassette:

• 3.0% CV at 300bp
• 9.0% CV at 1500bp

FAQs

The efficiency of recovery is somewhat dependent on the time of elution.  For minimum range cuts, >80% recovery is routine for  faster migrating fragments on a given cassette (100-200bp on a 2% gel, and 300-500 on a 1.5% gel). Recovery decreases as DNA size increases and a 50% yield is typical for the largest targets..

10μg of sheared genomic DNA is the recommended maximum per sample well.

Approx. 500ng of sheared genomic DNA is the least amount can be detected by the optical system. Without optical detection, cuts from 20-30ng samples have been reproducibly extracted. The lowest amount of DNA that can be loaded has not been fully evaluated.

High salt concentration, ligases, or other sample components can affect the mobility of DNA through agarose. The ionic strength of the sample should be lower than the ionic strength of the buffer (80mM monovalent ions). DNA binding proteins such as ligase are known to significantly change DNA mobility and should be removed.

Yes. Since the fractions are electro-eluted from agarose, there is very little residual low molecular weight contamination.

The instrument is equipped with fluorescence-based DNA detection optics which illuminate the Ethidium Bromide-bound DNA as it passes the detector.

Extractions are recovered in 40-60μl.

Extracted DNA is in buffer containing 51mM Tris, 28.8mM TAPS and 0.8mM EDTA, pH= 8.68. With ethidum bromide labelled casseetes, eluted sample contains 5μg/ml EtBr.

No, samples can be added directly to the amplification reactions.

It will typically take about 1-2 minutes per sample, or 7-8 minutes per gel cassette.

Run times vary depending on the size (bp) of the desired cut. See tables below for approximate total run times.  The run times shown are based on avereages; run can vary by ±5 minutes :