The BluePippin is a preparative electrophoresis platform that separates and extracts DNA fragments. Using pre-cast gel cassettes, DNA is automatically collected in buffer according to software-input size ranges. The BluePippin features pulsed-field electrophoresis capabilities that enable accurate collection of kilobase-sized fragments. Cassettes are available for DNA size selections between 50 bp and 50 kb.
There is NO fee to use the BluePippin for URI and RI-EPSCoR users.
System Overview. There are three components to the BluePippin system:
- Instrument – The instrument contains an epifluorescence detector, an electrophoresis power supply, an electrode array, and a Linux-based single-board computer. The system computer is accessed by an external LCD monitor, mouse, and keyboard. The electrode array is located in the top of the sliding instrument cover and is not user-accessible.
- Software – System software allows the user to enter parameters that define the DNA fragment size ranges or DNA bands that are to be collected. The software also logs fluorescence and electrical current data, which may be analyzed in a review screen.
- Gel Cassettes – Pre-cast, disposable agarose cassettes are manufactured by Sage Science. Cassettes are supplied in kits that contain 10 cassettes and sufficient reagents to complete all collections. Kits have been developed to address DNA size range collections at various size ranges, or DNA band capture.
- Four types of gel cassettes: 0.75%, 1.5%, 2.0% & 3.0% agarose
- Cassette capacity: 4-5 sample lanes with internal or external marker
- Load capacity: up to 10 µg with 200 ng optical sensitivity
- Pulsed-field electrophoresis collects fractions up to 50 kb
- Individual sample channels with collection chambers eliminate cross-contamination
- Automated collection of PCR bands or restriction fragments
- Provides narrow fragment size distributions
Collection Strategies. Cassette kits employ the following strategies:
For Size Selection of smaller DNA fragments (90 bp – 1.5 kb):
- Dye-free gels with labeled internal standards –Fluorescein labeled DNA internal standards are added to samples when run. The rates of migration of the internal standards are used to determine the collection timing within the sample lane. Up to five samples may be run per cassette.
For Size Selection of large DNA fragments (>2kb):
- Dye-free gels with labeled external marker – A fluorescein labeled DNA marker set is loaded in a dedicated sample lane during a run. The rates of migration of the markers are used to determine the collection timing the remaining sample lanes. Four samples and one DNA marker must be run per cassette.
For DNA Band Capture (>2 kb):
- DNA binding dyes with external standards — A DNA marker set is loaded in a dedicated sample lane during a run. A DNA binding dye (Midori Green) is loaded into the (+) buffer chambers of the separation channels of all sample lanes. During electrophoresis, the DNA binding dye enters the gel and provides fluorescent detection of the DNA bands and markers. The rates of migration of the markers are used to determine the band capture thresholds in the remaining sample lanes. Four samples and one DNA marker must be run per cassette. DNA binding dye must also be added by the user.
Proper sample loading is critical for best performance of BluePippin cassettes. For maximum reproducibility and accuracy, the sample should travel through the central section of the gel column, and should be bounded on all four sides by uniformly conductive media – either gel or electrophoresis buffer. The goal of the loading procedure is to produce this geometry in the sample loading well. Properly prepared samples will be 40 µl in total volume consisting of 30 µl of DNA mixed with 10 µl of BluePippin loading solution.
For detailed information consult the following references:
- Cassette Kit Reference Chart
- Quick Guide 3% Agarose Gel Cassette
- Quick Guide 1.5% Agarose Gel Cassette
- Quick Guide 0.75% Agarose Gel Cassette
- BluePippin Operations Manual