Pippin Prep : automated DNA size selection and collection
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
Size selection ranges
Several gel types are currently available. Cassettes are pre-cast and disposable. DNA is optically detected with ethidium bromide in band capture cassettes. Ethidium bromide-free gel cassettes are also available with pre-labeled DNA markers.
How the gel cassettes work
Gel Cassettes use either an external DNA size standard run in a dedicated sample lane or an internal standard to determine the timing of size-selected collections. With the first option, 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 are pre-cast with agarose gel and are disposable. Each cassette has 5 sample lanes (internal standard) or 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.
Narrow size range
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.
Minimum Size Range
2.0% Gel Cassette:
1.5% Gel Cassette:
What kind of yield/recovery can I expect in my extractions ?
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..
How much DNA can I run at a time ?
10μg of sheared genomic DNA is the recommended maximum per sample well.
What is the smallest amount of DNA that I can load ?
Approx. 500ng of sheared genomic DNA is the least amount can be detected by the optical system. Without optical detection, cuts from few nanograms samples have been reproducibly extracted. The lowest amount of DNA that can be loaded has not been fully evaluated.
How clean should my sample be prior to running on the Pippin Prep ?
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.
Can I use the Pippin Prep to remove unincorporated adapters ?
Yes. Since the fractions are electro-eluted from agarose, there is very little residual low molecular weight contamination.
How is the DNA visualized ?
The instrument is equipped with fluorescence-based DNA detection optics which illuminate the Ethidium Bromide-bound DNA or the pre-labeled DNA markers as they pass the detector.
What volume is my extracted DNA in ?
Extractions are recovered in 40μl or 25μl.
What buffer is my extracted DNA in? pH? EtBr ?
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.
Will my extracted sample inhibit amplification ?
No, samples can be added directly to the amplification reactions.
How long does it take to load a cassette and start a run ?
It will typically take about 1-2 minutes per sample, or 7-8 minutes per gel cassette.
How long does a run take ?
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 :
Ref : PIP0001
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