Navigation Links
Amplification of Mouse cDNAs for Microarrays Using the Eppendorf ,,, MasterTaq Kit

ssful PCR amplifications were verified by electrophoresis on agarose gels. PCR products were purified on a BioRobot 3000 (QIAGEN, Santa Clarita, CA) using the QIAquick 96 PCR BioRobot Kit. The yield of PCR amplicon was assayed using the Picogreen dsDNA quantitation kit (Molecular Probes) and a Bio-Tek FLX800 microplate fluorescence reader.

Preparation of fluorescently labeled cDNA
One g of polyA+ RNA (mouse spleen and liver, respectively) were converted into fluorescently labeled cDNA by incubation with an oligo dT primer (44 M final) at 70C for 10 minutes in a final volume of 9 l. The solution was then mixed with 11 l of a solution 1.8X first strand buffer, containing 18 mM DTT, 900 M dATP, 900 M dGTP, 900 M dTTP, 230 M dCTP, 230 M Cy3- or Cy5-dCTP (APBiotech, UK), 20 U of RNasin (Promega), and 200 U Superscript II reverse transcriptase (Life Technologies). The reaction was incubated at 42C for 2 hours and heated at 100C for 5 minutes. RNA was hydrolyzed by the addition of 1 l of 10 N NaOH and subsequent incubation at 37C for 15 minutes. The reaction was neutralized by the addition of 5 l of 1M Tris pH 7.2 and 2 l of 5N HCl, and cDNA probes were purified using a QIAquick PCR purification kit (Qiagen, Santa Clarita, CA). Typically, one-third of each reaction was used per hybridization.

Microarray fabrication, hybridization, washing, scanning, and data analysis
PCR products (1,152 in total) derived from the mouse Unigene set and control cDNA clones were printed on a reflective slide (APBiotech, UK). DNA clones were heat denatured for 3 minutes at 95C, chilled on ice, and arrayed using a Molecular Dynamics (Sunnyvale, CA) Generation III spotter.
'"/>

Source:


Page: All 1 2 3 4 5 6 7 8 9

Related biology technology :

1. prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification
2. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
3. Greater Amplification Specificity with New Hot Start PCR Enzyme
4. Enhanced Amplification of Long Targets with PfuTurbo DNA Polymerase
5. Optimizing pfuturbo DNA Polymerase Amplification Reactions with Perfect Match PCR Enhancer
6. Improve Amplification Specificity with Hot Start PCR Enzyme
7. Amplification of the amoA Gene of Nitrosococcus oceani Bacteria Using Eppendorf MasterTaq
8. Optimizing the Direct Amplification of Missing cDNA 5 Ends Using the Eppendorf Mastercycler gradient
9. Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler
10. Amplification of Epstein-Barr Virus Exon C with Difficult Primers Using Eppendorf Mastercycler gradient
11. Optimizing the Direct Amplification of Missing cDNA 5 Ends Using the Eppendorf Mastercycler gradient
Post Your Comments:
(Date:7/25/2014)... 25, 2014 WABC Radio show out of ... with Lorry Young will be hosting California-based Vet-Stem, Inc.’s Founder ... cell therapy in pets. Dr. Harman first visited the ... cell therapy for pets suffering from osteoarthritis and other degenerative ... Medicine industry forward. , Young has invited Dr. Harman back ...
(Date:7/25/2014)... July 25, 2014 /PRNewswire-iReach/ -- Xybion Corporation, the ... global corporations operating in highly regulated industries, announced ... annual Global User Group Conference, Xybion International Exchange, ... Philadelphia, Pennsylvania at the Sheraton Society ... This year,s conference theme ...
(Date:7/24/2014)... OAK RIDGE, Tenn., July 24, 2014 -- A novel ... at the Department of Energy,s Oak Ridge National Laboratory ... known for its unusual physical and electrochemical properties. ... examined how oxygen affects the surface of a perovskite ... electronic behavior. The new avenue to understand surface behavior ...
(Date:7/24/2014)...  ECC West Africa, LLC (ECC) signed a contract ... College Hospital (UCH) Ibadan Board of Management for the ... construct, and operate and maintain a new, state-of-the-art Cancer ... the first of the 4-phase project, ECC will conduct ... UCH Ibadan intends to finance and develop the Cancer ...
Breaking Biology Technology:Robert J. Harman, DVM, Founder and CEO of Vet-Stem, Inc. to Join New York Radio Host Lorry Young in Upcoming Episode of A “Paws” For Your Pet 2Robert J. Harman, DVM, Founder and CEO of Vet-Stem, Inc. to Join New York Radio Host Lorry Young in Upcoming Episode of A “Paws” For Your Pet 3Xybion Announces It Will Host International User Conference in Philadelphia 2Xybion Announces It Will Host International User Conference in Philadelphia 3ORNL study reveals new characteristics of complex oxide surfaces 2ECC Signs a Contract for the Finance, Design, and Construction of a Premier Cancer Institute in Ibadan, Nigeria 2
... ... is a thermally stable, economical and high-performance solution for operations with both high and ... ... heat transfer fluid is a thermally stable, economical and high-performance solution for operations with ...
... in the September 24th issue of Nature , ... technology to explore the ancient history of India, the ... reveals that nearly all Indians carry genomic contributions from ... groups experienced periods of genetic isolation from each other ...
... , ANN ARBOR, Mich., Sept. 23 ... Research Edition version 4.2, a significant product upgrade for ... has been fully committed to supporting academic and non-profit ... features a completely redesigned user-interface, improved search and filter ...
Cached Biology Technology:Paratherm's New GLT Heat Transfer Fluid: Thermally Stable & Lower Minimum Startup Temperatures 2New research reveals the ancestral populations of India and their relationships to modern groups 2New research reveals the ancestral populations of India and their relationships to modern groups 3Compendia Bioscience Announces the Release of Oncomine 4 Research Edition 2
(Date:7/25/2014)... characterized by the existence of numerous isoforms arising ... the protein deeply different characteristics. NRG1 plays an ... development and the different phases occurring after injury ... remyelination and target reinnervation, Researchers at the University ... NRG1 upregulation observed in Schwann cells immediately after ...
(Date:7/25/2014)... The routine use of a molecular testing panel ... performing the correct initial surgery for patients with ... University of Pittsburgh Cancer Institute (UPCI), partner with ... Multidisciplinary Thyroid Center and other diagnostic testing agencies, ... initial surgery by 30 percent, according to the ...
(Date:7/24/2014)... Keryx Biopharmaceuticals, Inc. (Nasdaq:KERX) (the "Company") announced the publication ... 3 study of Zerenex (ferric citrate), the Company,s investigational ... hyperphosphatemia in patients with end-stage renal disease (ESRD) on ... with FErric CiTrate in EsrD) was published online today ... Nephrology ( JASN ). , This ...
Breaking Biology News(10 mins):Test increases odds of correct surgery for thyroid cancer patients 2Test increases odds of correct surgery for thyroid cancer patients 3Zerenex™ (ferric citrate) long-term Phase 3 study results published in JASN 2Zerenex™ (ferric citrate) long-term Phase 3 study results published in JASN 3Zerenex™ (ferric citrate) long-term Phase 3 study results published in JASN 4
... sustains irreparable damage in the wake of a heart ... the cardiac muscle contains few stem cells, the tissue ... and cannot contract properly. In their search for ... exploring cardiac "patches" that could be transplanted into the ...
... The chemical is known as an "endocrine disruptor," a substance ... found in everything from plastic drink bottles to the linings ... for cash register receipts not to mention the urine ... BPA has been associated with the development of chronic diseases ...
... animal welfare concern and can cause great economic losses ... whole. A new website has been launched to help ... The website [ http://www.featherwel.org ] has been developed ... Veterinary Sciences to improve bird welfare through FeatherWel, a ...
Cached Biology News:A heart of gold 2BPA + chlorine = bad news 2New website will help farmers ensure hens maintain good feather cover 2
... Epithelial and endothelial cells-directed ... a RGD peptide-conjugated polyethylenimine which ... integrin-expressing epithelial and endothelial cells. ... in cell-cell and cell-matrix adhesion ...
... WinMelt software is used ... of any DNA sequence up ... in optimizing placement of primers ... detection by denaturing gradient gel ...
Request Info...
... To score SNPs, Polymorphic uses proven, ... standard in DNA analysis. We use high ... can be confirmed by sequencing in both ... provides sequence context information, further confirming the ...
Biology Products: