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Team:Bielefeld-Germany/Project/Background/Technology
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==HPLC== | ==HPLC== | ||
Revision as of 12:37, 21 September 2011
Contents |
PCR
The polymerase chain reaction (PCR) is a method to reproduce DNA in vitro. An enzyme, the polymerase, is used for this purpose. The term "chain reaction" in this context means the fact, that the products of previous cycles are used as template for the next cycle and thus exponential amplification of specific DNA fragments is achieved. The formed DNA product results from defined primers, which are the start point for the polymerase.
The PCR is used in biological and medical laboratories for a variety of different tasks. In our case we used the PCR for two different applications. It is possible to check successful cloning when using primers that bind at the prefix and suffix regions of the cloning plasmid and separate/analyze the PCR product by agarose gel electrophoresis (Colony PCR). The other task was the qualification of our molecular beacons. A thermal profile was taken by meassuring the fluorescence of the beacons as a function of temperature by a Real-time PCR device.
For the Colony PCR we used (device) and (device). The thermal profile assay was performed with the LightCycler® 480 System by Roche.
We used the PCR to characterize the following BioBricks: <partinfo>K525001</partinfo>; <partinfo>K525002</partinfo>; <partinfo>K525005</partinfo>; <partinfo>K525007</partinfo>; <partinfo>K525008</partinfo>; <partinfo>K525121</partinfo>; <partinfo>K525123</partinfo>; <partinfo>K525221</partinfo>; <partinfo>K525222</partinfo>; <partinfo>K525223</partinfo>; <partinfo>K525224</partinfo>; <partinfo>K525301</partinfo>; <partinfo>K525303</partinfo>; <partinfo>K525304</partinfo>; <partinfo>K525305</partinfo>; <partinfo>K525306</partinfo>; <partinfo>K525311</partinfo>; <partinfo>K525312</partinfo>; <partinfo>K525401</partinfo>; <partinfo>K525403</partinfo>; <partinfo>K525404</partinfo>; <partinfo>K525405</partinfo>; <partinfo>K525406</partinfo>; <partinfo>K525411</partinfo>; <partinfo>K525412</partinfo>; <partinfo>K525499</partinfo>; <partinfo>K525512</partinfo>; <partinfo>K525515</partinfo>; <partinfo>K525517</partinfo>; <partinfo>K525551</partinfo>; <partinfo>K525560</partinfo>; <partinfo>K525710</partinfo>; <partinfo>K525720</partinfo>; <partinfo>K525730</partinfo>; <partinfo>K525740</partinfo>; <partinfo>K525998</partinfo>; <partinfo>K525999</partinfo>
HPLC
High performance liquid chromatography (HPLC), a sort of liquid chromatography, is an analytical method in chemistry, not only to separates substances, but also to identify and quantify these substances by internal standards.
The examined substance, together with a mobile phase, is pumped through a separation column that contains the stationary phase. You can classify an HPLC according to the principle of separation in normal phase (NP), reverse phase (RP), ion exchange (IEC), size exclusion (SEC) and chiral chromatography. The substance to be tested is injected into the stream of the mobile phase in small amounts. The movement of the probe trough the column depends on the natures of the sample on the one hand and the stationary phase on the other hand. If one component of the tested substance interacts strongly with the stationary phase, it remains on the column relatively long. If the interaction of a substance is weak, its velocity to leave the column is higher. The time at which a specific substance elutes is called retention time and is suitable for identification under particular conditions.
The HPLC was performed with (device).
We used the HPLC to characterize the following BioBricks: <partinfo>K525512</partinfo>; <partinfo>K525515</partinfo>; <partinfo>K525517</partinfo>
MS/MS
Tandem mass spectrometry (MS/MS) is an analytical physics technique with multiple mass spectrometry sections combined. Between two analyzers, a collision cell is mounted to apply energy to the ions by colliding with an inert gas (usually nitrogen or argon). Thereafter the ions disintegrate very specific to other, lighter ions.
For the MS/MS analysis we used (device).
LC-MS
Liquid chromatography–mass spectrometry (LC-MS), a sort of tandem mass spectrometry, is an analytical chemistry technique which combines the physical separation of liquid chromatography (LC or HPLC) with the analysis of the substances by mass spectrometry (MS).
For the LC-ESI-qTOF-MS we had (device) available.
We used an LC-ESI-qTOF-MS to characterize the following BioBricks: <partinfo>K525512</partinfo>; <partinfo>K525515</partinfo>; <partinfo>K525517</partinfo>
MALDI-TOF
Matrix-assisted Laser Desorption/Ionization – Time of flight (MALDI-TOF) is a procedure to analyze large biomolecules by their mass.
Before measuring, the analyte is cocristalized on an metal plate within a solid matrix, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) and 2,5-dihydroxybenzoic acid (DHB) are most commonly used. Desorption of the analyte is taken by a laser beam, commonly with a wavelength of 337 nm. The matrix absorbs the laser light and the upper layer explosively vaporizes, ejecting both, matrix and analyte molecules. In addition to this vaporization, the laser beam also leads to an ionization of the analytes. The mass analysis is taken by the TOF (Time of flight)-Analysator. The evaporated ions are accelerated by an electric field. Typical acceleration voltages for this are 10-30 kV. The velocity of an ion depends on his mass and the charge. An ion detector converts the incoming ions into an electrical signal. The advantage of this method is the rapid analysis of a relatively large mass range.
The MALDI-TOF analysis was performed with (device).
We used the MALDI-TOF Analysis to characterize the following BioBricks: <partinfo>K525121</partinfo>; <partinfo>K525123</partinfo>; <partinfo>K525221</partinfo>; <partinfo>K525222</partinfo>; <partinfo>K525223</partinfo>; <partinfo>K525224</partinfo>; <partinfo>K525301</partinfo>; <partinfo>K525303</partinfo>; <partinfo>K525401</partinfo>; <partinfo>K525403</partinfo>
Fluorescence Assay
The fluorescence assay takes advantage of a molecules property to emit light of one specific wavelength while absorbing light of a different wavelength. This feature is given by a various amount of proteins, like GFP, RFP, etc. so that these peptides can be used as report proteins to detect other proteins by a fluorescence assay.
For the fluorescence assay we had the Infinite® M200 platereader by Tecan available.
We used the fluorescence assay tho characterize the following BioBricks: <partinfo>K525304</partinfo>; <partinfo>K525305</partinfo>; <partinfo>K525306</partinfo>; <partinfo>K525404</partinfo>; <partinfo>K525405</partinfo>; <partinfo>K525406</partinfo>
Luminescence Assay
The luminescence assay uses the ability of specific enzyme reactions to produce light for the measuring of product formation. On the other hand, this assay can also be used to detect enzymes or substrates which are required for these reactions.
For the luminescence assay we had the (device) available.
We used the Luminescence assay tho characterize the following BioBricks: <partinfo>K525311</partinfo>; <partinfo>K525411</partinfo>; <partinfo>K525999</partinfo>
FACS
Flow cytometry describes a measurement method, which allows the analysis of cells that past an electronic detection apparatus or a light beam at high speed. Depending on the shape, structure or coloration of the cells, different effects are generated, from which the properties of the cell can be derived.
In our case, we used the flow cytometer (device) to analyze the formation of our S-Layers on the surface of the silica beads.
We used the flow cytometry analysis to characterize the following BioBricks: BBa_K525301; BBa_K525303; BBa_K525304; BBa_K525305; BBa_K525306; BBa_K525311;
Microscopy
AFM
Atomic force microscopy (AFM) is a special type of scanning probe microscopy, with a gained resolution below a nanometer, a thousand times higher than the optical diffraction limit. During the measurement, a nanosized needle, fixed on a leaf spring, is performed across the surface of a probe. The leaf spring is bent in different amplitudes, depending on the surface structure of the sample. This bending or deflection of the spring can be measured with optical sensors and is a result of the atomic forces, occurring between the needle and the surface. By recording the deflections respective forces point by point, an image of the sample surface is generated like a digital photo while each pixel stands for a specific physical or chemical parameter. The possible resolution is determined by the radius of curvature of the spring leaf and ranges from 0.1 to 10 nm. Thus, the atomic force microscope reaches the highest resolution of any microscopic technique.
For the AFM we had the (device) available.
We used the AFM to characterize the following BioBricks: BBa_K525301; BBa_K525303; BBa_K525304; BBa_K525305; BBa_K525306; BBa_K525311
STM
Scanning tunneling microscopy (STM) is also a type of scanning probe microscopy, which differs in the configuration of the sensor and its interaction with the surface to be examined. In STM, the sensor is electrically conductive. The interactive process uses the concept of quantum tunneling. When a bias is applied between the tip and the surface, this leads to a measurable tunneling current as a function of the position of the sensor.
For the STM we had the (device) available.
We used the STM to characterize the following BioBricks: BBa_K525301; Baa_K525303; BBa_K525304; BBa_K525305; BBa_K525306; BBa_K525311
CLSM
Confocal laser scanning microscopy (CLSM) is a type of confocal microscopy, whereas, in contrast to the normal conventional light microscopy, not the entire sample is illuminated, but only a fraction, in most cases only a limited spot. The intensities of light reflected or emitted are measured consecutively at all locations of the sample, so that a subsequent construction of the image is possible.
For the CLSM we had the (device) available.
We used the CLSM to characterize the following BioBricks: BBa_K525301; Baa_K525303; BBa_K525304; BBa_K525305; BBa_K525306; BBa_K525311
References
H. Engelhardt (ed.): Practice of High Performance Liquid Chromatography - Applications, Equipment and Quantitative Analysis. Springer-Verlag, Berlin/Heidelberg/New York/Tokyo 1986, ISBN 3-540-12589-2.
M. H. Gey: Instrumentelle Analytik und Bioanalytik, 2. Aufl., Springer Verlag, Berlin 2008.
C. Hamann, M. Hietschold: Raster-Tunnel-Mikroskopie. Akademie Verlag, Berlin 1991, ISBN 3-05-501272-0.
Handbook of biological Confocal Microscopy, 3rd ed., Springer-Verlag, New York 2006, ISBN 978-0-387-25921-5
J. R. Lakowicz: Principles of fluorescence spectroscopy. Springer 2006, ISBN 9780387312781.
C. Mülhardt: Der Experimentator: Molekularbiologie / Genomics. 6. Aufl., Spektrum Akademischer Verlag, 2008, ISBN 978-3-8274-2036-7.
M. G. Ormerod (ed.): Flow Cytometry — A practical approach. 3rd edition, Oxford University Press, Oxford (UK.) 2000, ISBN 0199638241
J. Peter-Katalinic (ed.), F. Hillenkamp: MALDI MS: A Practical Guide to Instrumentation, Methods and Applications. Wiley-VCH, Weinheim 2007, ISBN 3-527-31440-7.
O. Shimomura: Bioluminescence: Chemical Principles and Methods. Word Scientific Publishing Company 2006, ISBN 981-256-801-8.
D. Skoog, J. Leary: Instrumentelle Analytik. Grundlagen, Geräte, Anwendung. Berlin 1996. (ger. translation of the 4th edition of Principles of Instrumental Analysis, Orlando 1992).
