WWW.ABSTRACT.DISLIB.INFO
FREE ELECTRONIC LIBRARY - Abstracts, online materials
 
<< HOME
CONTACTS



Pages:   || 2 | 3 | 4 |

«Table of Contents User manual part II: running the POSaM Platform Overview Slide Surface Preparation In-House Epoxysilane Slide Preparation ...»

-- [ Page 1 ] --

Table of Contents

User manual part II: running the POSaM Platform

Overview

Slide Surface Preparation

In-House Epoxysilane Slide Preparation

Materials:

Methods:

Commercial Slide Alternatives

Reagent Preparation

Overview:

Solvent Preparation

Materials:

Methods:

Phosphoramidites and Tetrazole

Materials:

Methods:

Base Deprotection Solution

Materials:

Methods:

Acetonitrile Wash Solvent

Materials:

Methods:

Oxidizer and Deprotection Acid

Materials:

Methods:

Arrayer Operation

Overview

Loading the Phosphoramidites and Tetrazole

Materials:

Method:

Preparing and Loading the Substrates

Materials:

Method:

Priming the Bulk Reagents

Method:

Testing and Clearing the Nozzles

Method:

Drying Down the Enclosure

Materials:

Method:

Pre-Washing the Slides

Method:

Initiating Synthesis

Method:

Post-Synthesis Processing

Overview

Cleanup

Materials:

Method:

Base Deprotection

Materials:

Method:

Shutting Down the Arrayer

Control Hybridization for Quality Control

Overview

Hybridization

Materials:

Post-Hybridization Washes





Materials:

Scanning

Materials:

Method:

Interpretation

Maintenance and Troubleshooting

Clearing Clogged Inkjet Pumps

Cleaning the Phosphoramidite and Tetrazole Supply Vials

Materials:

Method:

Changing the Inkjet Head

Materials:

Method:

System Properties

System Performance

Speed and Failure Rate

Spot Diameter

Hybridization Specificity

Two-color Hybridization

Cost of Operation

Future Improvements

Appendix A: INKJET ARRAYER STARTUP PROCEDURE CHECKLIST

User manual part II: running the POSaM Platform Overview Oligonucleotide synthesis has been an efficient processes since Itakura described the phosphotriester method of oligonucleotide synthesis in the mid 1970s. Reactivity, speed, coupling efficiencies, and recoveries have continued to improve, especially since the advent of automated oligonucleotide synthesizer, and much of the groundwork necessary for inkjet oligoarray synthesis had already been layed by these early investigations.

One problem, however, is that the preferred solvent for oligonucleotide synthesis, acetonitrile, is too volatile to work well during inkjet synthesis. Since the picoliter-size droplets are flying through the air at the speed of a flying bullet, the acetonitrile tends to evaporate so rapidly that sometimes it is gone even before it hits the slide surface. This high volatility results in frequent misplaced droplets, crystals of reagent sitting dry on the slide, and a very high rate of clogging the piezoelectric nozzles.

Blanchard and Hood suggested that the ideal inkjet solvent should have low volatility, a higher boiling point than acetonitrile, higher vapor pressure, be compatible with phosphoramidite synthesis, and not harm the inkjet array head. They eventually showed that propylene carbonate (PC) met these requirements and had a coupling efficiency close to that seen with acetonitrile.

The University of Washington patented propylene carbonate as a solvent for inkjet oligonucleotide synthesis, and granted Rosetta Inpharmatics (and then Agilent), exclusive rights to use propylene carbonate for oligoarray synthesis. To remain clear of patent infringements, we are have continued the search for the best solvent to use for inkjet oligoarray synthesis. We have found that a 1:1 mixture of 2-methyl glutaronitrile (MGN) and 3-methoxypropionitrile (3MP) works as well as PC, and appears to cause less nozzle clogging (unpublished observation).

If care is taken to follow the instructions outlined herein, and to maintain a scrupulously water free environment, labs building the POSaM platform should have little trouble constructing arrays. Selecting and printing the oligonucleotides best suited for the quantification of unique mynas, genes, or splicing sites is a different case, however, and will still require empirically testing different sequences to find those that discriminate best. As more people use the POSaM platform we will be able to build an annotated library of reporters known to identify specific mRNAs. Use of the POSaM platform for developing and testing novel array designs, developing empirical data on probe performance, and experimenting with new applications for microchip technologies should provide a stable, accessible, inexpensive tool for basic research laboratories Another critical factor is the surface chemistry of the solid substrate. We have tested a wide range of commercial substrates, and found that none of them can consistently deliver the balance of reactivity and hydrophobicity necessary to produce discrete, round, evenly-spaced features with an acceptable coupling efficiency to the slide surface. Although some commercial slides work reasonably well, in our hands the lot-to-lot, and even intra-lot, variations result in a very high failure rate (50%).

We developed a simple and effective method for surface-modifying our own slides, and have found this to be a much more stable and reliable approach. Protocols are presented in this manual for both the custom modification process and the use of commercial substrates, so that the end user may determine what is preferable in their situation.

Slide Surface Preparation In-House Epoxysilane Slide Preparation The critical balance of hydrophobicity and reactivity necessary for successful inkjet array synthesis is achieved by using a reactive epoxysilane capable of covalent attachment to the silanol groups on the glass surface in a background of longer-chain silanes which also attach to the glass but provide the hydrophobic properties and do not participate in base attachment during synthesis. These hydrophobic silanes are obtained by using RainX. Yes, RainX (the commercial treatment you use on your windshields to make rain bead up). We are currently in the process of testing the individual components to obtain the minimum necessary formulation and concentrations of silanes, so we are not tied to a specific commercial product with unknown purity and quality control. However, RainX is extremely cheap and has been incredibly effective in obtaining the correct surface properties.

Materials:

FisherFinest microscope slides Chemical fume hood 6-8 Glass slide staining dishes 4-6 glass slide staining racks Wire handle for staining racks, dedicated for nanostrip Wire handle for staining racks, dedicated for silanizing Nitrogen or Argon cylinder equipped with a blow gun P-1000 pipettor P-1000 compatible pipette tips 100ml pyrex graduated cylinder 250ml amber glass bottle Cap for 250ml bottle with either Teflon liner or Teflon-lined septa Medium-size sonicating water bath ( 4.5in x 6in x 4 in bath) Blotter material cut to fit into the base of a slide staining rack Clean plastic slide storage boxes sufficient for the number of slides.

Chemical Supplier Cat # Supplied Abbreviations 3-(glycidoxypropyl) Sigma-Aldrich 44016-7 100g bottle 3-GPTMS trimethoxysilane RainX Pennzoil Quaker-State RX11212 207ml bottle RainX Sodium Hydroxide Acros Organics 206060010 1kg bottle NaOH Hydrochloric Acid, Fisher Scientific A144-212 2.5L bottle HCl Concentrated (12M) Nanostrip Cyantek NC9096760 1 gallon Nano Ethanol, 200 proof Aaper Chemical n/a 1 gallon EtOH Acetone, HPLC-grade Fisher Scientific A949-4 4L bottle Acetone Methanol, HPLC- Fisher Scientific A452-4 4L bottle MeOH grade 18M-ohm Milli-Q wa- n/a n/a n/a 18MΩ H2O ter

–  –  –

Methods:

1) Fill several glass racks (usually 4-6) with 19 slides per rack, loaded in a zig-zag pattern with every other slide racked diagonally.

2) Fill 2 glass staining dishes 2/3 full with nanostrip. CAUTION: nanostrip is a concentrated acid mixture containing hydrogen peroxide. It is extremely aggressive against organic material, and should be considered very hazardous. It reacts exothermically with water, oxidizing agents, and organic solvents. It should be used only neat, never diluted or mixed with other chemicals. During storage, peroxides in nanostrip and waste materials can decompose, so it must always be stored in a vented bottle to prevent over pressurization and explosion.

Materials in contact with nanostrip must be clearly marked, and thoroughly rinsed with deionized water immediately after use. Any spills must be cleaned up immediately.

Nanostrip should only be used in a chemical fume hood, with appropriate protection against splashes, spills, and other exposure. Wearing nitrile gloves, goggles, and a laboratory coat should be considered the minimum protection; elbow-length rubber gloves, sleeve protectors, a face shield, and a chemical apron offer a higher degree of protection.



Pages:   || 2 | 3 | 4 |


Similar works:

«∋(9(/230(17 2) 6859(,//∃1&( 7(&+12/2∗ ∃1∋ 5,6. 2) ∃%86( 2) (&2120,&,1)250∃7,21 9ΡΟ  7ΚΗ ςΩ∆ΩΗ ΡΙ ΩΚΗ ∆ΥΩ ΛΘ ΦΡΠΠΞΘΛΦ∆ΩΛΡΘς,ΘΩΗΟΟΛϑΗΘΦΗ &20,17 ΡΙ ∆ΞΩΡΠ∆ΩΗΓ ΣΥΡΦΗςςΛΘϑ ΙΡΥ ΛΘΩΗΟΟΛϑΗΘΦΗ ΣΞΥΣΡςΗς ΡΙ ΛΘΩΗΥΦΗΣΩΗΓ ΕΥΡ∆ΓΕ∆ΘΓ ΠΞΟΩΛΟ∆ΘϑΞ∆ϑΗ ΟΗ∆ςΗΓ ΡΥ ΦΡΠΠΡΘ Φ∆ΥΥΛΗΥ ς∴ςΩΗΠς ∆ΘΓ ΛΩς...»

«DATE: December 21, 2012 TO: PJM RPM Auction Participants SUBJECT: Frequently Asked Questions on RPM/ACR and RPM Must Offer Obligation Monitoring Analytics, LLC serves as the Market Monitoring Unit (MMU) for PJM Interconnection, L.L.C. and is also known as the Independent Market Monitor (IMM) for PJM. The following are frequently asked questions and answers related to its role in the PJM Reliability Pricing Model (RPM) Capacity Market. For questions related to this document, please email...»

«KARIN BÜCHTER, DIETMAR FROMMBERGER & H.-HUGO KREMER (Helmut-Schmidt-Universität Hamburg, Universität Magdeburg & Universität Paderborn) EDITORIAL zur Ausgabe 23: Akademisierung der Berufsbildung In der (berufs-)bildungspolitischen Diskussion gehört die Akademisierung der Berufsbildung zu einer der zentralen Forderungen. Zu verstehen sind unter dieser Thematik zwei Blickrichtungen. Einerseits wird die Frage aufgeworfen, inwieweit in der beruflichen Bildung und auf der Basis beruflicher...»

«Miniature radio frequency transponder technology suitability as threatened species tags SCIENCE FOR CONSERVATION: 71 Murray E. Douglas Published by Department of Conservation P.O. Box 10-420 Wellington, New Zealand Science for Conservation presents the results of investigations by DoC staff, and by contracted science providers outside the Department of Conservation. Publications in this series are internally and externally peer reviewed  January 1998, Department of Conservation ISSN...»

«STATE FARM MUTUAL FUND TRUST Legacy Legacy Institutional Class A Class A Class B Class B Class Class R-1 Class R-2 Class R-3 Ticker Ticker Ticker Ticker Ticker Ticker Ticker Ticker Symbol Symbol Symbol Symbol Symbol Symbol Symbol Symbol State Farm Equity Fund SNEAX SLEAX SNEBX SLEBX SLEIX SREOX SRETX SREHX State Farm Small/Mid Cap Equity Fund SSNAX SFSAX SSNBX SFSBX SFEIX RSEOX RSETX RSEHX State Farm International Equity Fund SNIAX SFFAX SNIBX SFFBX SFIIX RIEOX RIETX RIEHX State Farm S&P 500®...»

«SUPERINTENDENT PERCEPTIONS OF THEIR PROFESSIONAL DEVELOPMENT IN LEADERSHIP FOR STUDENT ACHIEVEMENT AT TEXAS REGIONAL EDUCATION SERVICE CENTERS A Dissertation Presented to The Faculty of the College of Graduate Studies Lamar University In Partial Fulfillment of the Requirements for the Degree Doctor of Education in Educational Leadership by Jerry G. Maze May 2009 SUPERINTENDENT PERCEPTIONS OF THEIR PROFESSIONAL DEVELOPMENT IN LEADERSHIP FOR STUDENT ACHIEVEMENT AT TEXAS REGIONAL EDUCATION SERVICE...»

«Information & Security: An International Journal Predrag Tasevski, vol.32, 2015 http://dx.doi.org/10.11610/isij.3204 MACEDONIAN PATH TOWARDS CYBERSECURITY Predrag TASEVSKI Abstract: Information and communication technologies in Macedonia have experienced a phenomenal growth throughout the last decades, which has had a tremendous impact on governmental services’ presence in the Internet, as well as on everyday life. Against this background, technologies-based growth introduces new risks and...»

«1 A s if conjuring my dream, the earth shook. My throat closed, and I knew I should run, or duck, or roll—any one of the things I’d been taught since elementary school to do during an earthquake. But I stood rooted, watching the horses gallop up from the creek that bordered the redwood forest—their hooves striking the cracked ground as they flew, their tails raised high, their necks extended, their ears flattened against their heads, desperate to keep up with their herd mates. A cloud of...»

«Please read this document carefully as the terms below apply to your use of the Season Ticket In particular, we ask you to note the following: The Club may in any Season introduce a scheme by which each Ticket Holder’s Season Ticket would automatically renew for the subsequent Season. If the Club does intend to introduce an Automatic Renewal Scheme it would inform each Ticket Holder that it intends to implement such scheme and each Ticket Holder would be provided with the option to be...»

«Australasian Academy of Paediatric Dentistry President: Prof Nigel King Vice President: Dr Nina Vasan Secretary: Dr Fiona Bell Treasurer: Dr Peter Wong Minutes of AAPD 19th Annual General Meeting 3rd April 2013, Melbourne, Australia Meeting commenced 14.20 1. Welcome: Professor Nigel King 2. Present: Eduardo Alcaino, Mary Apps, Fiona Bell, Angus Cameron, Wendy Cheung, Helen Cornwell, Mala Desai, Prashanth Dhanpal, Rebecca Eggers, Daniel Ford, Peter Gregory, Roger Hall, Sally Hibbert, Tim...»

«SEKTOR PSIKOLOGI DAN KAUNSELING JABATAN PENDIDIKAN NEGERI PERAK ( THE VIRTUE OF PATIENCE ) Kesabaran ialah sifat yang dipuji oleh semua agama dan diakui kebaikannya oleh para ilmuan, raja dan pentadbir. Orang yang sabar menerima segala cabaran dengan senang, memikirkannya dengan teratur dan jika timbul masalah dia tidak berputus asa. Semangat ketabahan yang ada pada seseorang yang sabar itu menunjukkan kewibawaannya dalam menjalankan tugas, keberaniannya menghadapi dugaan dan kepercayaannya...»

«LYON x GeNeVe x SHANGHAi Annexe du Rapport Scientifique AcAdemic YeAr 2007-2008 EDUCATING ENTREPRENEURS FOR THE WORLD www.em-lyon.com Annexe au Rapport Scientifique Annuaire de la Recherche 09/2007 08/2008 Sommaire Articles académiques Articles de presse Ouvrages Chapitres d’ouvrages Cas publiés Cahiers de recherche Thèses soutenues Thèses dirigées Interviews de la Faculté Communications en conférences Annexe au Rapport Scientifique Annuaire de la Recherche 09/2007 08/2008 Articles...»





 
<<  HOME   |    CONTACTS
2017 www.abstract.dislib.info - Abstracts, online materials

Materials of this site are available for review, all rights belong to their respective owners.
If you do not agree with the fact that your material is placed on this site, please, email us, we will within 1-2 business days delete him.