Navigation Links
Quantitation of DNA for Automated Sequencing Using the VersaFluor Fluorometer

Wenda Ribeiro and Margaret Saha, Department of Biology, College of William and Mary, Williamsburg, Virginia 23187


Introduction
DNA sequence analysis is an integral part of genetic research. Recently, the use of automated sequencers has become increasingly more common in small research laboratory settings. However, the success of automated long read sequencing reactions require precise picomolar concentrations of DNA. We have successfully employed the VersaFluor fluorometer (Bio-Rad) to accurately and routinely quantify DNA for sequencing with the LiCor 4200 automated sequencer.


Materials and Methods
A genomic clone for xHif (Xenopus hypoxia inducible factor) was isolated by a homology screen of a Xenopus muscle genomic library using a 32P-dCTP labeled mouse probe (a gift of Steven McKnight). Phage DNA was isolated using a standard protocol, digested with EcoRI and HindIII and run on a 1.2% agarose gel (Figure 1). Bands corresponding to insert DNA were excised and the DNA isolated on glass milk. The DNA fragments were subcloned into BlueScript SK+ (Stratagene) and transformed into DH5a cells. Plasmid DNA was isolated using anion exchange chromatography. A λZap (Stratagene) cDNA clone for Xenopus xHif was also isolated using a homology screen of a tadpole library. Rescued plasmid DNA was obtained using the manufacturers instructions and was subsequently transformed into DH5α cells. Plasmid DNA was isolated using anion exchange chromatography.

Quantitation of the DNA was achieved using the Fluorescent DNA Quantitation Kit (Bio-Rad) with a 360 nm excitation filter and a 460 nm emission filter. The standard curve was established for quantification of DNA concentrations ranging from 1005,000 g/ml by plotting seven points of varying DNA concentrations made by serial dilution of control calf thymus DNA. All dilutions were made in 2 ml of 1 g/ml Hoechst 33258 dye and included 0 ng DNA as a blank and 10,000 ng DNA as the upper range. The values were recorded in relative fluorescence units (RFUs) and were plotted on a graph with the x axis = DNA concentration (ng) and the y axis = RFU (Figure 2). The slope of the line was determined to be 1.0.

By employing the equation y = mx+b, we established the value of b to be equal to 0. Therefore, by setting the upper range value at 10,000 RFU using 10 l of 1 mg/ml calf thymus DNA and using 2 l of the sample DNA in 2 ml of the 1 g/ml Hoechst dye, the RFU reading is equal to ng/ml for our sample. We are then able to calculate the volume of DNA needed to obtain a 250 femtamolar concentration per reaction.


Results and Discussion
Using the Excel II sequencing kit (Epicenter) and quantifying our DNA with the VersaFluor fluorometer, we have been able to achieve highly readable and reproducible sequencing runs of up to 1,200 bp with the LiCor 4200 automated sequencer. This has enabled us to sequence the entire 2.2 Kb cDNA for Xenopus hypoxia inducible factor, xHIF, in several runs, and will allow us to sequence our genomic clone easily and efficiently.

The correct concentration of DNA in each reaction is the key to obtaining good reads on the LiCor 4200. This entails precise quantitation of the DNA. The VersaFluor fluorometer has proven to be easy to calibrate and provides excellent and reproducible results.

This work is supported by National Institute of Health Grant 1815HD32080 and National Science Foundation Grant 1BN9553176.


back to top
'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Quantitation of Cabergoline at Extremely Low Plasma Concentrations with a Triple Quadrupole Mass Spectrometer
2. Bioanalytical Method Intraday Validation for the Quantitation of Paroxetine in Bovine Plasma using the TSQ Quantum Mass Spectrometer
3. Quantitation of the Protein MIA as a Marker for Chondrocytes in Research Samples
4. Comparison of Different Methods of mRNA Quantitation
5. Quantitation of RNA for Hybridization Blots Using the VersaFluor Fluorometer
6. Quantitation of Lymphangiogenesis Using the iCycler iQ Real-Time PCR Detection System and Scorpions Detection System, Rev A
7. Quantitation of GAD67 Gene Expression in Prefrontal Cortex of Schizophrenia Patients Using the iCycler iQ Detection System and Molecular Beacons, Rev A
8. Benchmark Plus Microplate Reader: Quantitation of Protein Concentration Using Two Different Colorimetric Assay Kits, Rev A
9. A Robust LC/MS/MS Method for the Quantitation of Very Long Chain Fatty Acids for Research into the Characterization of Peroxisomal Disorders
10. A new LC/MS/MS Research Method for Rapid Quantitation of Five Immunosuppressant Drugs, including Mycophenolic Acid (MPA)
11. Separation and Quantitation of R and S Enantiomers using Normal Phase Chromatography on an API 3000 LC/MS/MS System
Post Your Comments:
(Date:7/21/2014)... (PRWEB) July 21, 2014 ... Workforce Investment Board (WIB) and Las Positas College ... provide technical education and hands-on training for veterans, ... Technology Program at Las Positas is designed ... needed for engineering technician careers, and establishes a ...
(Date:7/21/2014)... , July 21, 2014  Mike Patterson, CEO ... $1.6 million in Series Seed B funding to ... sensors for the life science, defense, intelligence, and ... Partners led the round and was joined by ... Said Patterson , "This round of funding ...
(Date:7/19/2014)... United States Non-Vascular Stents Market ... report, "United States Non-Vascular Stents Market Outlook to ... States Non-Vascular Stents market. The report provides value, ... and average prices (in US dollars) within market ... Metal Biliary Stents (Metal Non-Covered Biliary Stents, Metal ...
(Date:7/18/2014)... Reflecting the importance of the evolution of Houston’s real estate ... founder and president of Customized Real Estate Services , ... approach to the firm’s branding. , According to Ms. ... of his or her commercial clients have grown exponentially over ... in this market to fully embrace the potential for helping ...
Breaking Biology Technology:Engineering Program Established for Veterans 2Engineering Program Established for Veterans 3Global Graphene Manufacturing Leader, Graphene Frontiers, Receives Series Seed B Funding To Assure Production Of The Next Generation Of Biological And Chemical Sensor Technology 2United States Non-Vascular Stents Market Outlook to 2020 | Researchmoz 2United States Non-Vascular Stents Market Outlook to 2020 | Researchmoz 3United States Non-Vascular Stents Market Outlook to 2020 | Researchmoz 4LEED AP Certified ITRA Global / Customized Real Estate Services Introduces New Approach to Its Branding 2
... Contributed by James V. Anderson and Charles L. Guy, The,University of Florida, Institute ... Introduction , ... , Glucose Regulated Proteins) are ER-luminal proteins ... 1 BiPs are associated with the transport and folding process , ...
... Contributed by Alan T. Bakalinsky Department of Food Science and Technology, ... , Abstract , ... cerevisiae were , compared to those of ... supported previous genetic findings that two of the wine strains are aneuploid , ...
... Marie Nguyen and William Strong, Bio-Rad Laboratories, Inc.,6000 James ... , Introduction , ... is an established technique employed for protein separation. However, ... manual steps and several , hours to ...
Cached Biology Technology:A Rapid Method for the Purification of Analytical Grade Proteins From Plants Using Preparative SDS-PAGE and Preparative IEF 2A Rapid Method for the Purification of Analytical Grade Proteins From Plants Using Preparative SDS-PAGE and Preparative IEF 3A Rapid Method for the Purification of Analytical Grade Proteins From Plants Using Preparative SDS-PAGE and Preparative IEF 4A Rapid Method for the Purification of Analytical Grade Proteins From Plants Using Preparative SDS-PAGE and Preparative IEF 5Electrophoretic Karyotypes of Wine Strains of Saccharomyces cerevisiae 2Electrophoretic Karyotypes of Wine Strains of Saccharomyces cerevisiae 3Electrophoretic Karyotypes of Wine Strains of Saccharomyces cerevisiae 4Electrophoretic Karyotypes of Wine Strains of Saccharomyces cerevisiae 5Electrophoretic Karyotypes of Wine Strains of Saccharomyces cerevisiae 6Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A 2Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A 3Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A 4Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A 5Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A 6Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A 7Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A 8Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A 9Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A 10Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A 11Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A 12Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A 13
(Date:7/21/2014)... Their scruffy beards weren,t ironic, but there are reasons ... the Ice Age. , According to research from the ... liked living in Greater Cincinnati long before it was ... age. A study led by Brooke Crowley, an assistant ... enjoyed the area so much they likely were year-round ...
(Date:7/21/2014)... July 21, 2014 Dysfunction in dopamine signaling ... genes in the brain,s prefrontal cortex and may ... disorders, such as schizophrenia, according to UC Irvine ... in brain cells that receive this neurotransmitter showed ... affect a variety of behavioral and physiological functions ...
(Date:7/21/2014)... and Aqua satellites detected 154 hotspots in areas across ... land fires had increased again following a decline in ... report was far higher than what had been reported ... , The hotspots were scattered in six regencies and ... areas. Smoke and the related haze it creates could ...
Breaking Biology News(10 mins):Mammoth and mastodon behavior was less roam, more stay at home 2Mammoth and mastodon behavior was less roam, more stay at home 3UCI researchers find epigenetic tie to neuropsychiatric disorders 2
... State University and the University of Chicago has uncovered clues ... N4, injects an unusual substance -- an RNA polymerase protein ... The results, which are published in the current issue of ... of the infection strategies used by viruses that attack bacterial ...
... as an homage to achievement in the field of coral ... link between corals of the Pacific and Atlantic. Dr. Ann ... McNeill of the University of Miami named a new species ... some six million years old after renowned coral reef ...
... was managing several ecosystem research projects on Galveston Island when the 2008 hurricane season began in June 2008. Then ... Click here for more information. , ... , , , , ... , , , , ...
Cached Biology News:Researchers study virus with unusual properties 2Researchers study virus with unusual properties 3Isopora or isn't it? 2Dune and dirty: Hurricane teaches lessons through ecosystem research 2Dune and dirty: Hurricane teaches lessons through ecosystem research 3Dune and dirty: Hurricane teaches lessons through ecosystem research 4Dune and dirty: Hurricane teaches lessons through ecosystem research 5Dune and dirty: Hurricane teaches lessons through ecosystem research 6
... offers the most comprehensive, cost-effective solution ... have incorporated unprecedented quality into both ... includes proven detection chemistries and antibodies ... TechMate protocol. It also includes our ...
Human Cell Line Slides...
Mouse Cell Line Slides...
Request Info...
Biology Products: