Services
– BluePippin (SageScience)
The BluePippin (jpg) has the capabilities of the Pippin Prep, plus pulsed-field electrophoresis for resolving and collecting high molecular weight DNA. Target sizes or ranges of sizes are entered in software, and fractions are collected in buffer with no possibility of cross contamination in the cassette. Gel cassettes for DNA selections are available in 4 agarose concentrations to provide a full range of size selection options between 100bp up to 50kb. Run times range from ~30 minutes (small fragments) to several hours (pulsed-field runs for very large DNA fragments). Visit SageScience's website for product specifications of its BluePippin kits.
BluePippin Cassette kits
Kits contain 10 disposable pre-cast gel cassettes. All necessary standards (i.e., Markers) and reagents are included in the kit; however, Gel Cassettes and Markers can be "mix-&-match" by purchasing Marker separately. Cassettes that use internal markers have 5 samples lanes; cassettes using external markers have 4 sample lanes. Reimbursement is "per lane used". Before submitting a request for size-selection by BluePippin cassettes, please ask if the desired kit is in stock; the most common kit stocked by the Core is in bold below.
1) Size-selection Breakpoints: Despite the explicit values that can be input for size-selection, results may differ by up to ~50bp from intended. Thus, do not expect your size-selected product to precisely match the values used in the run protocol – instead, broaden the requested size range to accomodate the expected offsets.
2) Kit Specifications: Choosing and running a Bluepippin kit is something more akin to an art than a science. Results really depend on the combination of Gel Cassette, Marker, and Cassette Definition used. Further, while Kits contain specific Markers, all Markers can be purchased separately from kits -- so, mix-&-matching of cassettes and Markers is possible.
Thus,
Example: Assume the Core has the BLF7510 kit (0.75% gel cassette; Marker S1) in stock and two clients have different size selection needs for high molecular weight fragments:
– Client 'A' wishes to collect all fragments 20 kb and above.
– Client 'B' needs to eliminate both fragments <20kb and fragments larger than some value under 50kb.
For Client 'A', perform a 20-kb High Pass collection.
- Kit: BLF7510 (0.75% Cassette and Marker S1).
- Cassette Definition: 0.75%DF Marker S1 High Pass 15-20 kb.
- Range mode: bp start = 20,000bp; bp end = 50,000 bp.
- External Marker S1 (note S1 degrades rapidly after expiration, so non-expired S1 is recommended).
- Kit: BLF7510 (0.75% Cassette) – or, the BRT7510 kit which already contains External Marker E).
- Cassette Definition: 0.75%DF 9-30kb R+T Marker E2.
- Range+T mode: Bp start= 20,000bp; elution time: depends on the desired end collection size.
- External Marker E2: separate purchase required as the BLF7510 kit contains External Marker S1.
– For Client 'A', the specified combination will collect all fragments 20-kb and larger. Sage Science reports having been able to collect a 165kb band using each of these High-Pass cassette definitions.
–For Client 'B', the specified combination will eliminate fragments smaller than 20kb as well as those larger than the specified high end.
Online Submission Request
- Form: Make all necessary 'selections', including the 'check box', and click 'submit'. Please note that "
Quantity " for this assay refers to the number oflanes required to process all the samples. - Size-Selection: Depending on cassette type and targeted sizes, collected fragment sizes (as determined on the Bioanalyzer) may differ from expectations by up to 5-15% (for details, see BluePippin Cassette Specifications). Further, we have seen that the size ranges collected on the 1.5% gels tend to run smaller than expected, while they run larger than expected on the 2.0% gels; these biases must be taken into account if you need to avoid a specific fragment size.
- Product yield: Maximum yields are obtained with broad vs. tight size selections. If needed, following collection of the DNA from the elution modules, additional yield may be obtained by collecting buffer added to the emptied elution modules (albeit, at the cost of reduced product concentrations).
- Sample-related information: In the "Client Memo" box, provide all information necessary to process your samples (e.g.: target size for Tight collections vs. minimum/maximum fragment size for Range collection; maximize recovery (see below); and "special requests").
- Special Requests: We might be able to adjust protocols to handle cases where your desired size-selections conflict with the normal protocols. For example, suppose you wish to target 150-600bp using the 2%-gel cassettes, which normally co-migrate two marker bands (50-bp, 150-bp) with the sample in each cassette lane (i.e., they use 'internal' markers). However, by sacrificing a sample lane, we can run the Marker solution as an "external marker", thereby allowing us to start collecting your DNA at 150-bp in the other 4 lanes. If your sample requires special handling, please contact the Core to discuss the situation and then provide the necessary information in the 'Comment' field for your online submission.
- Maximum Recovery: Typically, a few µl of sample remain in the elution module after collecting the elution buffer. If you request "Maximum Recovery", 40 µl of TVLE (10 mM Tris, 0.05 mM EDTA) will be added to the elution well, mixed by pipetting, and collected. In this case, you will receive ~70 µ/sample vs. the normal ~35 µl/sample. Naturally, the DNA concentration will be considerably reduced and the buffer concentration (see below) will be altered. If necessary, since the second collection is done with TVLE, you can subsequently restore the DNA concentration by means of the SpeedVac.
- Processed Samples: The electrophoresis buffer is 50 mM Tris, 30 mM TAPS, and 0.1 mM EDTA. However, as EDTA does not rapidly equilibrate across the ultrafiltration membrane of the elution module, the final EDTA concentration of eluted samples is elevated to ~1-2 mM. Of course, in 'maximum recovery' situations, the buffer concentrations are lower.
Physical Submission of Samples
- Submission #;
- Date;
- PI's Last Name; and,
- Submitter name or initials.
