ABI Prism 6100 Nucleic Acid PrepStation

Transcription

1 Product Bulletin Sample Preparation Systems ABI Prism 6100 Nucleic Acid PrepStation I solation of highly pure nucleic acids free of cross contamination,for results you can count on utomation of key processes ensures A easy high-throughput purification ersatile format purifies up to 96 V samples of total RNA or genomic DNA samples from a variety of biological sources S mall footprint saves valuable laboratory bench space The ABI Prism 6100 Nucleic Acid PrepStation provides high-throughput nucleic acid preparation within your budget. Introduction Isolation of nucleic acids from biological materials has traditionally been very labor-intensive, prone to errors and often yielding impure and inconsistent products. Fast, small, versatile, and within your budget, the ABI Prism 6100 Nucleic Acid PrepStation is a stand-alone nucleic acid purification platform that isolates and purifies total RNA and genomic DNA from a variety of biological samples. A typical total RNA isolation from 96 samples of a cell culture requires only 30 minutes. Quality Purification Through Integrated Vacuum Systems and Precise Electronic Control The 6100 PrepStation contains integrated vacuum system under precise electronic control and a series of uniquely designed hardware features. When combined with newly developed chemistries, these features enable run-to-run and well-to-well high-yield purification that is reproducible, and free of cross contamination. The purification station carriage holds the application-specific, 96-well purification tray. Cellular lysates or homogenized tissue lysates are added to the tray and

2 vacuum is applied under user interface control for a specific time and at a defined pressure. The vacuum s waste position directs cellular debris and wash solutions to an external 4-L reservoir, while a collection position elutes the purified nucleic acid in one of two formats. The first format is the industry standard, 96-well PCR microplate, while the second is a 96-well, 1-mL deep-well plate. The tray and other consumables used on the 6100 PrepStation are manufactured under stringent conditions and are guaranteed to be free of contaminating DNA, RNA, and RNase activity, giving you confidence in the basic elements of your research. Easy to Learn, Simple to Operate No computer is required to run the 6100 PrepStation, but an integrated serial port connection allows easy installation of future software upgrades. The 6100 PrepStation was designed with several innovative features to make it easy to operate for even the most inexperienced member of your Figure 1. Rat Liver Figure 2. Rat Lung Rat Liver A 260/280 Ratio Rat Lung A 260/280 Ratio A 260/280 Ratio A 260/280 Ratio Rat Liver Replicate Samples Rat Lung Replicate Samples Rat Liver µg RNA/well Rat Lung µg RNA/well 4 1 µg RNA per well (A 260 O.D.) µg RNA per well (A 260 O.D.) Rat Liver Replicate Samples Rat Lung Replicate Samples Figure 1 through 5. Eight samples of each tissue were homogenized in 1x total RNA lysis buffer: 1 volume of 2x total RNA lysis reagent (P/N ) plus 1 volume of phosphate buffered saline (PBS) using either a Polytron rotor/stator homogenizor (animal tissue) or a Waring blender (plant tissue). 800 μl of each homogenized tissue lysate was filtered through the tissue pre-filter tray (p/n ). 500 μl per well of each filtered and homogenized tissue lysate was purified on the ABI Prism 6100 PrepStation. For the 1% agarose gel images, approximately 2 μg of purified total RNA was added to each lane. Yields and purity were measured using a UV spectrometer. The following concentrations of materials were purified on the total RNA purification tray (p/n ) after filtration using the tissue pre-filter tray. Rat Liver: 20 mg/ml of 1x lysis buffer Rat Lung: 10 mg/ml Rat Heart: 5 mg/ml Rat Brain: 100 mg/ml Corn Leaf: 200 mg/ml

3 laboratory. The graphical user interface is an intuitive scrolling menu, similar to that of a thermal cycler, that provides easy access to pre-programmed methods so you can create, edit, and store custom purification routines. The instrument is controlled with a simple four-direction keypad and five soft-key positions. The Quick Run feature and preprogrammed methods for a variety of applications make the instrument simple to learn. A liquid crystal display provides a real-time view and full details about each run, including the status of the vacuum system, current commands, pre-programmed methods, user identification, and diagnostic tools. A printout of all 6100 instrument methods can be generated, including a run log. The Run Log feature enables Quick Runs for new routines to be readily stored as new methods for future use. The Run Log also keeps track of the vacuum position (either waste or collection ), set point, and achieved values. Figure 3. Rat Heart Figure 4. Rat Brain Rat Heart A 260/280 Ratio Rat Brain A 260/280 Ratio A 260/280 Ratio A 260/280 Ratio Rat Heart Replicate Samples Rat Brain Replicate Samples Rat Heart µg RNA/well Rat Brain µg RNA/well 40 µg RNA per well (A 260 O.D.) µg RNA per well (A 260 O.D.) Rat Heart Replicate Samples Rat Brain Replicate Samples

4 To get you up and running, we include an interactive training CD that guides you through each step of the 6100 PrepStation s setup from removing the instrument from its packaging to completing a purification run. The CD will acquaint you with all the basic operations of the 6100 PrepStation. Video footage and diagrams guide you through purification using the Quick Run feature. Flexible Enough For All Your Future Demands Those with more experience will find it easy to create, edit, and store their own customized methods. The system accommodates methods from up to 20 members of the laboratory staff and 300 methods total, each of which can contain up to 99 steps. PIN numbers assure privacy, and each method is stored and cross-referenced by name, date, and method for easy retrieval. Should your laboratory require even higher throughput with walk-away automation in the future, methods developed for the 6100 PrepStation are completely compatible with, and directly transferable to, the ABI Prism 6700 Automated Nucleic Acid Workstation. Figure 5. Corn Leaf Figure 6. Human Blood Corn Leaf A 260/280 Ratio Human Blood (CPD) Total RNA A 260/280 Ratio A 260/280 Ratio A 260/280 Ratio Corn Leaf Replicate Samples CPD Replicate Samples Corn Leaf µg RNA/well Human Blood (CPD) Total RNA µg/ml Blood µg RNA per well (A 260 O.D.) µg RNA per ml blood (A 260 O.D.) Corn Leaf Replicate Samples CPD Blood Replicate Samples Figure 6. Whole Human Blood. To 1 volume of freshly drawn (never frozen) blood, 1 volume of PBS was added, followed by an equal volume of total RNA lysis reagent (P/N ). 500 μl of this lysate was added to the total RNA purification tray (P/N ), and the 6100 PrepStation vacuum passed the lysate across the purification tray membrane. This process was repeated four times, giving a total of 2 ml of lysate per well added to the tray before washing and elution of purified total RNA.

5 Threshold Cycle (CT) Well Position Figure 7. Well-to-Well Cross-Contamination Assay. Twenty-three 200-μL samples containing 1 x 10 6 copies of an RNA template were spiked into a 6100 Prepstation for purification. Water was added to the remaining 73 wells, and full purification for total RNA was performed, generating a 96-well output plate. Then a one-step RT-PCR 5 nuclease-based assay using TaqMan probes was created for each purified well using the GAPDH amplicon. Blank Wells CT=40 Spike Wells CT=17 In this assay, a cross-contamination event is considered to have occurred if the CT difference between a blank and a spiked well is less than 20 CT units. The results show that the purification process results in no well-to-well contamination. (A decrease in CT value for a blank well would signify cross-contamination.) Figure 8. Total RNA Chemistry Reagents.

6 Instrument System Components ABI Prism 6100 Nucleic Acid PrepStation Integrated vacuum pump Liquid crystal display Directional key pad 5 soft-key positions 4-L waste reservoir with integrated pump-protecting filter Training CD Optional printer Pre-programmed methods for total RNA purification from cultured cells, plant and animal tissues, and whole blood Pre-programmed methods for genomic DNA from plant and animal tissues Dimensions ABI Prism 6100 Nucleic Acid PrepStation Width 53 cm (21 in) Depth 48 cm (19 in) Height 27 cm (10.5 in) Weight 20.5 kg (45 lbs) ORDERING INFORMATION Description Quantity Part Number ABI Prism 6100 Nucleic Acid PrepStation Starter Kit of Reagents and Disposables as listed below Reagents and Disposables Lysis reagent, total RNA 250 ml Wash solution 1 1 L Wash solution 2 1 L Elution solution Total RNA purification trays 10/box TCII reaction plate, 96-well 20/box Deep-well plates 10/box Splash guards 20/box For Research Use Only. Not for use in diagnostic procedures. Notice to Purchaser: License Disclaimer The ABI Prism 6100 Nucleic Acid PrepStation conveys no patent rights, expressly or by implication, under any patent or patent application owned by or licensable by Applera Corporation that covers any thermal cycling instrument, apparatus or system, any composition, reagent, or kit, or any process. Specifically, but without limitation, no right, immunity, authorization, or license is granted, expressly or by implication, for the processes of reverse-transcription PCR or the 5 nuclease assay Applied Biosystems. All rights reserved. Applied Biosystems, AB (Design), and ABI Prism are registered trademarks and Applera is a trademark of Applera Corporation or its subsidiaries in the US and/or certain other countries.taqman is a registered trademark of Roche Molecular Systems, Inc.All other trademarks are the property of their respective owners. Printed in the USA, 10/2006 Publication 117PB04-02 Headquarters 850 Lincoln Centre Drive Foster City, CA USA Phone Toll Free International Sales For our office locations please call the division headquarters or refer to our Web site at