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Missouri S&T cDNA RESOURCE CENTER1. What is the Missouri S&T cDNA Resource Center?
2. What is the rationale for the DNA Resource Center?
3. How is the Center funded?
4. How are the clones obtained?
5. What vector are the clones in?
6. How do I order the clones?
7. How are the clones shipped?
8. What documentation is provided?
9. How do I "reconstitute" the cDNA after it arrives?
10. What are the restrictions on the use of the clones?
11. Can I give the clones to other investigators?
12. How should I acknowledge the source of the clones in manuscripts?
13. Who do I contact regarding shipping and/or technical issues?
14. I ordered a stable cell line. What do I do now?
15. What's with the Biological Science Department at Rolla? 1. What is the Missouri S&T cDNA Resource Center? The Missouri S&T cDNA Resource Center is a service provided by the faculty of the Department of Biological Sciences of Missouri University of Science and Technology.
The purpose of the cDNA Resource Center is to further scientific investigation by providing cDNA clones of human proteins involved in signal transduction processes. The goal of the Center is to accelerate scientific discovery in human biology and pathology. This is achieved by providing high quality clones for important signaling proteins in a timely manner.
By high quality, we mean that the clones are: - Sequence verified
- Propagated in a versatile vector (pcDNA3.1+)
- Free of extraneous 3' and 5' untranslated regions
- Available in wild-type, epitope-tagged and common mutant forms (e.g., constitutively-active or dominant negative)
- Expression-verified in most cases by coupled in vitro transcription/translation assays
By timely, we mean that the clones are - Most orders shipped within one business day by courier delivery (Federal Express).
We expect this service to allow scientists to focus on basic biological questions without spending excessive amounts of time in procuring, preparing or verifying their own (or their colleagues) cDNA clones. We expect these clones to be particularly useful to scientists probing and reconstructing signal transduction pathways in model systems.
Back 2. What is the rationale for the Missouri S&T cDNA Resource Center? Biological research often involves the reconstruction of signaling pathways. The basic tools required in this work are the clones of the relevant interacting proteins. We found that we were spending inordinate amounts of time obtaining, verifying and modifying these clones. Accordingly, we felt that research would be accelerated and a great deal of duplicate effort eliminated if there was a service that provided rapid access to high quality sequence-verified clones.
We offer human clones because of their obvious inherent interest. We remove the untranslated regions because their presence often increases variability in expression. We utilize a standard vector to simplify handling procedures.
Back 3. How is the Center funded? A $120 fee is charged to cover our costs for producing, verifying, and distributing the clones.
Back 4. How are the clones obtained? Usually the polymerase chain reaction (PCR) is used to amplify the ORF from commercially prepared cDNA. The PCR product is then sub-cloned into a plasmid vector (see below) and then sequence verified against published sequences. Discrepancies, if any, from the published sequence(s) are noted in the clone documentation. In the case of novel sequences, such as previously undescribed human orthologs, the predicted translation product is compared with amino acid sequence of orthologous proteins from other species. GenBank submissions are prepared to describe these data.
We have also obtained clones from investigators who wish to deposit their clones in the Resource Center (e.g., WOX1, AGS1). The ORFs of deposited clones are sub-cloned into the standard vector and the sequence of the insert verified. We thus maintain a standard "context" for each clone in the collection.
Back 5. What vector are the clones in? Clones are distributed by agreement in Invitrogen's pcDNA3.1+ vector. This vector can be used for mammalian expression (CMV promoter, bovine growth hormone polyadenylation signal), production of sense strand RNA by in vitro transcription (T7), production of single stranded DNA (f1 origin), and production of stably transfected cell lines (neomycin resistance). The vector contains the ampicillin resistance gene and can be propagated in common E. coli strains. (www.invitrogen.com)
Please note that pcDNA3.1 is the property of the Invitrogen Corporation, Carlsbad, CA. In consideration for these materials, you agree that you will not, without prior written permission from Invitrogen: a) use that vector for any commercial purpose; b) amplify, duplicate, clone or otherwise use the vector or any part thereof independently of the gene insert provided; c) transfer the vector or any part thereof (except the gene insert) to third parties.
Back 6. How do I order the clones? Clones can be ordered via our web page (www.cdna.org), by FAX (573 341-7609) or by email at cdna@mst.edu. We accept payment by purchase order (P.O.) numbers, wire transfers, MasterCard or Visa. We will mail an invoice to customers paying by P.O. or wire transfer. Please be aware, if using a Bank Transfer for payment, that the purchaser is responsible for any and all bank transfer fees.
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7. How are the clones shipped? Most orders received are shipped within one day by Federal Express; overnight for orders within the U.S., or by international priority service for international orders. The clones are provided as dried DNA (approximately 5.0 ug) within a 0.5ml screw-cap tube. Instructions for reconstituting the DNA are provided below.
Back 8. What documentation is provided? To save on processing costs, only an packing slip is shipped with the clones. For each clone, up to four forms of documentation can be downloaded from our web site: 1) an information sheet that contains details on the insert, including how the clone was obtained, any sequence discrepancies, and experimental mutagenesis details, if applicable; 2) a sequence sheet that contains the nucleotide sequence of the insert and the predicted translation product; 3) a text file in FASTA format containing the insert nucleotide sequence can be downloaded for use with your molecular biology software; 4) for the relevant clones a PDF file documenting expression in a coupled transcription/translation assay system. PDF files can be downloaded and viewed with Adobe Acrobat Reader (www.adobe.com).
Back 9. How do I "reconstitute" the cDNA after it arrives? Store clones at 4C until you are ready to use them. Clones are supplied as dried DNA (approximately 5.0 ug)within a 0.5ml screw-cap tube. Centrifuge the tube BEFORE OPENING to ensure pellet is at the bottom of the tube. Add 50-100ul of ddH20 or TE buffer and vortex or flick the tube to ensure that the pellet disolves.
Use 1-2 ul of supernatant for use in transfecting E. coli by electroporation or chemical means. Please do not try to use the DNA directly for any application other than to transform bacteria and prepare a large stock.
Back 10. What are the restrictions on the use of the clones? The clones are supplied solely for research purposes. Details on use of the material are included on the Material Transfer Agreement that is available on this web site. Please also note the restrictions on the use of the pcDNA3.1+ (see FAQ question #5), which is the property of the Invitrogen Corporation.
Back 11. Can I give the clones to other investigators? We would prefer that you do not for two reasons. First, for quality control purposes we would prefer to handle distribution of the clones ourselves. Although we cannot guarantee the suitability of the clones for a given research purpose, we will investigate, and correct, if appropriate, any sequence discrepancy from that provided in the documentation. Second, the future funding for the Missouri S&T cDNA Resource is contingent upon our meeting certain benchmarks for utilization. The two most important benchmarks are the number of clones we ship and citation of the resource in scientific manuscripts (see below). Hence, it is important that we quantify the number of clones that are supplied by the Resource and have a record of the laboratories using the clones to justify further development and maintenance of this service. All users of the clones should be aware of and agree to all points of the Material Transfer Agreement.
Back 12. How should I acknowledge the source of the clones in manuscripts?
Scientific communications describing work utilizing clones or associated material provided by the Missouri S&T cDNA Resource Center Material should acknowledge this source, referencing our Web Site. For example: "The cDNA clone for human Go-alpha was obtained from the Missouri S&T cDNA Resource Center (www.cdna.org)."
Back 13. Who do I contact regarding shipping and/or technical issues? To reach us by FAX use (573) 341-7609 or you may email (cdna@mst.edu).
Order/Payment Questions: cDNA Resource Center staff members are available at (573) 341-7610 on Monday through Friday between 9:00AM-2:00 PM, CST. or via email.
Technical Assistance: Email is the preferred method for technical issues, or you may also call our Lab Manager directly at (573) 341-6988. Requests will receive a response within one business day.
Back 14. I ordered a stable cell line. What do I do now? Delivery & Storage: CHO cell lines are shipped on dry ice but must be removed from the shipping container immediately upon arrival and either reconstituted (see below) or stored in liquid nitrogen (vapor phase) until needed.
Reconstitution: Thaw the vial @ 37 degrees celcius and slowly add 5-10ml of growth medium. Centrifuge briefly to re-pellet the cells. Remove supernatant and re-suspend in fresh growth media. Each vial contains 1-2 million cells and should be plated accordingly to achieve a 50-80% confluency overnight. Split cells the following day or every 3-4 days following general cell culture guidlines.
Back 15. What is the Department of Biological Sciences at Rolla? The Department of Biological Sciences Department of the Missouri University of Science and Technology sponsors the cDNA Resource Center. The department is a component of the Missouri S&T College of Arts and Sciences, and has 10 faculty members. The department has approximately 125 undergraduate majors and 20 graduate students in Applied Environmental Biology.
The Missouri S&T Biological Sciences Program is highly focused on molecular and cellular biology. The department is distinguished by particularly strong interactions with engineering and physical science programs.
For over 130 years, the Missouri University of Science and Technology (Missouri S&T) has been one of the nation's premier universities for science, technology, and, of course, engineering. Missouri S&T was founded in 1870 as the Missouri School of Mines and Metallurgy (MSM). Missouri S&T was the first technological university west of the Mississippi and one of the first in the United States. In 1964, MSM became known as the University of Missouri-Rolla, then in 2008 we became Missouri S&T. The Missouri University of Science and Technology now awards undergraduate and graduate degrees in 35 fields of engineering, science, humanities, and social sciences. Missouri S&T ranks among the top 25 American universities in the number of undergraduate engineering degrees awarded each year.
Faculty, Department of Biological Sciences
Missouri University of Science and Technology
105 Schrenk Hall, Rolla, MO 65409
campus.mst.edu/biosci
573-341-7274
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