Eppendorf Centrifuge 5424 Service Manual

[Cris Luczak]

TheUNLV Genomics Core Facility offers full service Affymetrix GeneChip microarray processing of Gene 1.0 ST expression arrays. We also offer processing of RNA extractions and have a bioinformatics specialist on site for data analysis. The UNLV Genomics Core Facility is capable of processing 3'IVT, Exon, miRNA, SNP, Tiling and other Affymetrix array types. Because microarrays are an individualized service, please contact the UNLV Genomics Core Facility Manager Casey Hall-Wheeler to discuss your specific needs.

For Gene and Exon expression array services, the user provides total RNA sample and user provides the chip. Listed prices include: front-end QC analysis of RNA sample using Agilent Bioanalyzer, two-cycle cDNA synthesis, labeling, and Affymetrix GeneChip Instrument System for hybridization, washing, staining and scanning.

For more information, including consultation for fluorochrome combination feasibility, necessary controls prior to designing your experiment, obtaining protocols, and scheduling flow cytometry, please contact the UNLV Genomics Core Facility Flow Cytometry Technician, Casey Hall-Wheeler.

Rate for full service flow cytometry = $45/hour. Selected flow cytometry reagents available at cost. We currently provide hundreds of single or multicolor conjugated antibodies for pilot studies at no charge.

The Agilent 2100 Bioanalyzer utilizes automated gel electrophoresis and microfluidics technology to separate, stain, destain, detect, image and analyze RNA, DNA and protein samples. This platform can be used to determine the size, quantity and quality of RNA, DNA and protein as well as two color flow cytometry of cells. This instrument offers a wide range of applications, including:

The UNLV Genomics Core Facility currently offers full service RNA analysis using the Agilent RNA Nano 6000 reagents and chips. These chips have the capacity to run up to 12 RNA samples per chip, require 1 l input volume, have a quantitative range of 25-500 ng/l and a qualitative range of 5-500 ng/l.

We also offer full service for all other Agilent RNA, DNA, protein and flow cytometry chips. Please contact the UNLV Genomics Core Facility Manager Casey Hall-Wheeler for pricing on all other Bioanalyzer applications.

The UNLV Genomics Core Facility offers Sanger DNA sequencing services. Sanger sequencing is run using an either an ABI 3130 or ABI 3500 Dx genetic analyzer and ABI BigDye v3.1 Terminator sequencing chemistry. The typical contiguous read length is > 900 bp, with QV's >20 for high quality, clean DNA templates. The typical turn-around time is two to four business days, although this may change depending on our sample workload.

To submit samples for sequencing, please provide the appropriate amount of DNA template (see the chart below) and primer (3.2 pmol to 10 pmol) in 0.2 ml flat-cap PCR tubes (no strip tubes please) in a final volume of 10 ul of nuclease-free water. Please do not submit templates in TE buffer for the EDTA will chelate the metal ions (Mg++) required for the cycle sequencing reaction.

Submitted samples must be clearly labeled in 0.2 ml flat-cap PCR tubes and each sample must have a unique identifier. The requestor must complete a DNA Sequencing Sample Submission Form and send it by email to Casey Hall-Wheeler at: casey...@unlv.edu before bringing sequencing samples to the Genomics Core.

Measures must be taken by the investigator to provide the cleanest DNA template possible for the overall success of the sequencing reaction and data is highly dependent on the quality and quantity of the DNA sample. A pGEM 3Zf(+) control is run with every sample set to ensure instrument and technical performance. Unincorporated dye terminators are removed from the extension products with using the BigDye XTerminator Purification Kit. We can use specialized cycle sequencing and genetic analyzer run protocols for difficult to sequence templates including short PCR products, GC-rich templates, or sequencing primers with non-standard annealing temperatures (inquire for details).

We run sequencing Monday through Thursday by scheduled appointment only. Our turn around time is between two to four days from the time you submit your samples until you have data in hand, although this timing is dependent on our current workload. To schedule an appointment for DNA sequencing services, please call Casey Hall-Wheeler at the UNLV Genomics Core at 702-895-1057.

The fee for DNA sequencing for samples that are ready to run and do not need to be manipulated by us is $6.25 per sequencing reaction. The fee for DNA sequencing for samples that need to be manipulated by us (addition of primer, verify template concentration on the Nanodrop, transfer submitted samples that are not in 0.2 ml flat-cap PCR tubes into 0.2 ml flat-cap PCR tubes) is $7.50 per sequencing reaction. For a complete list of our DNA sequencing pricing, please see our Fees page.

The UNLV Genomics Core Facility offers full service sample processing using the Qubit 3.0 Fluorometer. The Qubit 3.0 Fluorometer accurately measures dsDNA, oligos, RNA, microRNA, and protein at low concentrations, using as little as 1 uL of sample. The Qubit 3.0 is more sensitive than UV absorbance, as it uses highly sensitive Qubit quantitation assays containing fluorescent dyes that emit a signal only when bound to the target molecule. This specificity minimizes the effects of contaminants such as free nucleotides, intact/degraded DNA and RNA, protein, and excess salts. The Qubit 3.0 is ideal to quantify samples used in downstream applications such as quantitative real-time PCR or next-gen sequencing. The Qubit 3.0 can also be used as a mini-fluorometer by selecting either the blue LED excitation source with emission in the green and far-red channels, or the red LED excitation source with emission the far-red channel, to generate RFU values in the 300 - 1,000 nm range.

The rate for full service Qubit 3.0 Fluorometer sample processing = $3.00/sample, which includes the Qubit reagents, consumables, sample processing, and electronic data delivery. Alternatively, labs who wish to quantify their own samples can purchase their own Qubit Assay Kit and can use the Qubit 3.0 Fluorometer free of charge.

The Qubit 3.0 Fluorometer accurately measures dsDNA, oligos, RNA, microRNA, and protein at low concentrations, using as little as 1 uL of sample. The Qubit 3.0 is more sensitive than UV absorbance, as it uses highly sensitive Qubit quantitation assays containing fluorescent dyes that emit a signal only when bound to the target molecule. This specificity minimizes the effects of contaminants such as free nucleotides, intact/degraded DNA and RNA, protein, and excess salts. The Qubit 3.0 is ideal to quantify samples used in downstream applications such as quantitative real-time PCR or next-gen sequencing. The Qubit 3.0 can also be used as a mini-fluorometer to generate RFU values in the 300 - 1,000 nm range by selecting either the blue LED excitation source with emission in the green and far-red channels, or the red LED excitation source with emission the far-red channel.

The Amersham Typhoon 5 Biomolecular Imager is a versatile scanner that can image multi-fluorescent, radioisotope-labeled, phosphorescent, chemiluminescent, and colorimetric samples on gels, membranes, phosphor screens, multi-well plates, culture dishes, glass slides, and tissue sections. Fluorescence scans using 5 laser excitation sources, blue 488nm; green 532nm; red 635nm; IR short 685nm; IR long 785nm, facilitate multiplexing and visualization of all visible and IR fluorescently-conjugated antibodies and labels. Accurate quantitation with signal detection as low as 3 pg of protein and differences across a dynamic range with greater than five orders of magnitude. Pixel resolution as low as 10 m produces high-resolution images. The large scanning area of 40 46 cm supports high sample throughput, enabling simultaneous imaging of up to 20 gels or blots measuring 10 8 cm in size, or up to 9 multi-well plates in a single scan. The UNLV Genomics Core offers IQTL software for image analysis of relative band intensity and purity analysis of gels/blots, colony counting, and relative fluorescence in plates.

The DigiLab Genomic Solutions OmniGrid 100 Microarrayer is a high capacity multi-axis microarrayer that prints DNA samples onto solid substrates. This instrument is fully automated and is capable of handling of up to 72 input plates and holds 100 standard 1" x 3" glass slides. This instrument possesses a 48-pin head that can print in excess of 100,000 features per slide with a resolution of 2.5 m on the X and Y-axis and 1.25 m on the Z-axis. The samples are spotted onto the slides in patterns of rows and columns known as sample groups that can be adjusted according to personal preference.

The GenePix 4000B Scanner can be used to scan DNA and protein microarrays as well as tissue and cell arrays. This scanner has dual lasers that can acquire data at wavelengths of 532 nm (Cy 3) and 635 nm (Cy 5) simultaneously. This instrument scans standard 1" x 3" glass slides and produces images with a resolution range of 5 m to 100 m. GenePix Pro software is available for microarray image acquisition and data analysis, which uses industry standard GAL and GPR file formats.

The Nanodrop 1000 is a full spectrum spectrophotometer (UV and visible spectrum, 220-750 nm) that can measure the absorbance of DNA, RNA, protein and dyes using as little as 1 l sample volume without the use of cuvettes or capillaries. This instrument is routinely used to quantify and check the purity of RNA and DNA before proceeding with PCR or microarray assays.

The SpectraMax Plus384 is a full spectrum spectrophotometer (190-1000 nm) which contains both a cuvette port and a microplate drawer. Any standard cuvette, 12 x 75 mm test tube, 96 or 384-well microplate can be used to obtain absorbance measurements.

The Beckman Coulter Avanti J-E centrifuge is a refrigerated floor model preparative centrifuge. The micro-processor controlled Avanti J-E can be programmed to run at temperatures between 40C and -10C. Available rotors include the JA-25.50 rotor with a maximum speed of 21,000 rpm, and the JA-10 rotor with a maximum speed of 10,000 rpm.

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