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:10/1/2014)... 30, 2014 This report analyzes ... US$ Million by the following Product Segments: ... Specific end-use segments also analyzed are Pharmaceuticals ... The report provides separate comprehensive analytics for ... East, and Latin America. Annual estimates and ...
(Date:9/30/2014)... news release is available in German . ... the Technische Universitt Mnchen (TUM) have demonstrated a new ... experiments show that future computer chips could be based ... As the main enabling technology of the semiconductor industry ... limits, the TUM researchers and collaborators at the University ...
(Date:9/30/2014)... Shimadzu Scientific Instruments has released ... highly precise tool for measuring displacement in rubber ... 1000-mm maximum movement distance, the DSES-1000 can measure ... length of 10 mm. In addition, it achieves ... stroke and within +/- 100 µm below 50-mm ...
(Date:9/30/2014)... Jose, California (PRWEB) September 30, 2014 ... measure the level of each type of cholesterol carrier ... since the presence of excessive cholesterol levels in the ... a precursor condition to heart attack and other cardiovascular ... fatality worldwide, therefore testing for cholesterol levels on a ...
Breaking Biology Technology:Global Specialty Enzymes Industry 2Global Specialty Enzymes Industry 3Global Specialty Enzymes Industry 4Global Specialty Enzymes Industry 5Global Specialty Enzymes Industry 6Global Specialty Enzymes Industry 7Global Specialty Enzymes Industry 8Global Specialty Enzymes Industry 9Global Specialty Enzymes Industry 10Global Specialty Enzymes Industry 11Global Specialty Enzymes Industry 12Global Specialty Enzymes Industry 13Global Specialty Enzymes Industry 14Global Specialty Enzymes Industry 15Global Specialty Enzymes Industry 16Global Specialty Enzymes Industry 17Global Specialty Enzymes Industry 18Global Specialty Enzymes Industry 19Global Specialty Enzymes Industry 20Global Specialty Enzymes Industry 21Global Specialty Enzymes Industry 22Global Specialty Enzymes Industry 23Global Specialty Enzymes Industry 24Global Specialty Enzymes Industry 25Global Specialty Enzymes Industry 26A new dimension for integrated circuits: 3-D nanomagnetic logic 2A new dimension for integrated circuits: 3-D nanomagnetic logic 3Shimadzu’s New Long-Travel Extensometer Increases Precision for Soft Material Testing 2Surging Diabetic, Obese and Hypertensive Population Drives the Global Cholesterol Testing Market, According to New Report by Global Industry Analysts, Inc. 2Surging Diabetic, Obese and Hypertensive Population Drives the Global Cholesterol Testing Market, According to New Report by Global Industry Analysts, Inc. 3Surging Diabetic, Obese and Hypertensive Population Drives the Global Cholesterol Testing Market, According to New Report by Global Industry Analysts, Inc. 4
... Lund University in Sweden have discovered a new stem cell ... several different cell types -- most importantly, they can form ... finding to develop methods to heal and repair disease and ... biopsies, the researchers for the first time found stem cells ...
... April 19, 2012  Phigenics LLC, an innovative water management ... paper that demonstrates up to 33% false-positive test results ... methods. A false-positive result means the test indicated ... detectable levels when in fact it was below detectable ...
... Biofuels, LLC, a joint venture between BP and ... collaboration with Fagen, Inc. for the introduction of ... the world,s most experienced biofuels engineering, procurement and ... any other company.  They are the largest, most ...
Cached Biology Technology:New Stem Cell Found in the Brain 2New Stem Cell Found in the Brain 3Phigenics Research Challenges Accuracy Of Conventional Legionella Testing Protocol 2Phigenics Research Challenges Accuracy Of Conventional Legionella Testing Protocol 3Butamax™ and Fagen Announce Collaboration to Retrofit Ethanol Plants for Biobutanol Production 2
(Date:9/30/2014)... Researchers of Ludwig-Maximilians-Universitaet (LMU) in Munich report that ... cells more sensitive to chemotherapeutic drugs. They have ... a new target for anti-tumor agents. , ... Professor Stephan Sieber of the Technische Universitt Mnchen ... a potential new weapon in the fight against ...
(Date:9/30/2014)... 2014  Spectra Automation, a trusted supplier ... and power generation industries, announced today the ... an easy-to-use and cost-effective data collection, analysis ... laboratories. Most upstream process ... systems. Accessing and consolidating data from these ...
(Date:9/29/2014)... of gut bacteria called Clostridium ramosum , coupled with ... work is published this week in mBio , the ... , A research team from the German Institute of Human ... bacteria including C. ramosum gained weight when fed ... ramosum were less obese even when consuming a high-fat ...
Breaking Biology News(10 mins):Cancer therapy: Driving cancer cells to suicide 2Spectra Automation Introduces RECONN Bioprocess Manager 2Gut bacteria promote obesity in mice 2
... deposition associated with industry and agriculture can drive soils ... and polluting surface waters, according to a new study ... study, conducted in the Tatra Mountains of Slovakia by ... of Sciences, and the U.S. Geological Survey, shows what ...
... both physically and mentally, but genetically everyone is ... for decades now. But with population research becoming ... Tim Caulfield is concerned that genetic research could ... Nobel Prize winning geneticist James Watson claimed there ...
... new study by researchers at UC Davis Medical Center ... patients may be a result of their brains not ... drops in blood oxygen levels are more common than ... senior author Masud Seyal, a professor of neurology at ...
Cached Biology News:Acid soils in Slovakia tell somber tale 2Could genetic research awaken racist attitudes? 2Drops in blood oxygen levels may be key to sudden death in some epilepsy patients 2Drops in blood oxygen levels may be key to sudden death in some epilepsy patients 3
Mouse monoclonal antibody raised against a partial recombinant TPR. NCBI Entrez Gene ID = TPR...
... sample is a complex protein sample, ... when used with ReadyPrep 2-D starter ... sample can be used as a ... and protocol before using more difficult ...
... materials for 1,464 array sample elements. , ,Includes: ... , Blocker BSA in PBS (10X): 50 ml ... g , BupH Phosphate Buffered Saline: ... 2 x 10 ml ampules , SuperSignal West Pico ...
... Mouse polyclonal antibody raised ... Immunogen: ... 112 a.a) partial recombinant protein ... Accession Number: NM_130777 ...
Biology Products: