BioMicroCenter:PippinPrep

HOME --

SEQUENCING --

LIBRARY PREP --

HIGH-THROUGHPUT --

COMPUTING --

DATA MANAGEMENT --

OTHER TECHNOLOGY

The BioMicroCenter utilizes the Sage Scientific Blue Pippin ;http://www.sagescience.com/wp-content/uploads/2016/09/BluePippin-Operations-Manual-460013-Rev-D.pdf] for any gel based sizing. The instrument can be run as a walk-up or as an assisted service as part of Illumina, PacBio, or Oxford Nanopore library prep.

Preparative Gel Electrophoresis

Instrument Pippin/BluePippin TYPE WALKUP or ASSISTED assisted service available as part of library preparation UNIT Per run for walkup Per lane for assisted FORMAT 5 lane gel from 0.75% agarose to 3% agarose INPUT >1ug of DNA in low salt buffer EXPECTED YIELD 10-50% of material in the selected size range Narrower ranges result in lower yield. WALKUP SIGNUP ilabs calendar NEW USERS New users should request training by emailing biomicro@mit.edu DONATED BY Department of Biology	The BluePippin (Sage Science) is an automated preparative gel electrophoresis system that simplifies size fractionation of DNA. Runs of the five lane gels take approximately 1 hour to overnight depending on the fragment length and can either use internal/internal lane markers for calibration or ethidium bromide to detect passing bands. The BluePippin can separate bands between 50bp and 50kb. We have done some testing of the BluePippin system and the results are HERE. Critically, the system does require large amounts of input. Yields of <50% are typical within the desired range so multiple ug of input are strongly recommended before using the Pippin systems. Samples with lower amounts of input should consider alternative approaches including SPRIbead cuts. The system is based on precast gel cartridges. These gels narrow as they run, concentrating the sample from the initial 40ul (30ul sample + 10ul loading buffer) loaded in the wells. The BluePippin uses an optical detector to measure the rate of passage of bands based on high speed fluorescent markers or by directly detecting the DNA using ethidium bromide. Once the desired band passes the detector, the valve on each lane switches from a waste reservoir to an elution chamber. Once the band is passed, the valve turns back to waste and the desired band can be removed from the elution chamber with a pippette. Samples can be cleaned up with either SPRI beads or using an ethanol precipitation.	BluePippin BluePippin Gel
The BluePippin has four different percentages of agarose gels that can be used for different size selections. A stock of most types of cartridges are kept in the BioMicro Center. For long fragments using the pulse field, there are multiple settings on the BluePippin, each optimized for a different size range.		GEL SIZE RANGE NOTES 3.0% 50-200bp Used for small RNA libraries 2.0% 100-600bp 1.5% 0.3-1.5kb Used for RNAseq libraries 0.75% 1.0-50kb Uses pulse field.