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Figure 3: Spectrum of Plasmid - DNA
Figure 4. Melting curve of Plasmid - DNA
Figure 5. Melting point of Plasmid - DNA
Figure 6. Melting curve of thymus - DNA
Figure 7. Melting point of thymus - DNA
Native DNA melts in general at high temperatures often above 85°C like the melting point of human DNA which is at 86°C. The melting point of synthetic DNA can be estimated according to the base composition and the chain length.
The lower melting point of the Plasmid DNA is a result of the short chain length of the molecule having 48.5 base pairs (bp). The thymus DNA is composed more complex having a molecular weight of 13 kilobase pairs (kbp). Therefore the melting point of the thymus DNA is correspondingly higher.
Furthermore the melting point of nucleic acids depends on other factors like the chemical composition of formed hybrids, the salt concentration of the buffer solution as well as the specific reagents that are given to destabilise the DNA. Besides the higher proportions of mismatches of the base pairs the lower the melting point will be.
References
[1] Peter Yakovchuk, Ekaterina Protozanova and Maxim D. Frank-Kamenetskii. Base-stacking and base-pairing contributions into thermal stability of the DNA double helix. Nucleic Acids Research 2006 [2] Allgemeine Mikrobiologie, Georg Fuchs, Hans Günther Schlegel, 8. Auflage, Thieme Verlag [3] Genetik, Jochen Graw, 4. Auflage, Springer Verlag Berlin Heidelberg, 2006 [4] SPECORD®
PLUS manual, Analytik Jena [5] SPECORD® PLUS accessory manual, Analytik Jena
Summary
Nucleic acids store life’s blueprint in their special molecular structure. The hyperchromic effect, which is caused by a temperature increase separating the DNA double-strands, allows the DNA melting point to be determined by UV spectroscopy. The SPECORD®
PLUS, in combination with peltier temperated
accessories, allows the highly precise determination of melting points. Depending on the DNA material and the applicative requirements, temperatures can be programmed over a wide range and in different measurement modes. Use of an ultra-microcell makes it possible to work with microlitre sample volumes. A temperature sensor in the cell exactly monitors the sample temperature and makes sure that it is always equal to the programmed temperature and heating delays do not occur. With this configuration a temperature accuracy of ± 0.1°C is attained.
New IR Imaging Application
Syngene announced that its new range of G:BOX Chemi advanced multi-application image analysers can be used for imaging with infra red (IR) LI-COR IRDye®
dyes, making it easier to detect and quantify different types of proteins on multiplex Western blots.
The G:BOX Chemi systems, when fitted with a combination of recommended lighting and specific Syngene filters can be used for imaging LI-COR dyes, IRDye®
680 (Epi Red Multiplex LED lighting module and Syngene 705M filter) and IRDye® 800 (Epi LED IR 740
lighting module and Syngene LY800 filter). The GeneSys software in the G:BOX Chemi automatically selects the right lighting and filters for whichever IR dye or other fluorescent dyes scientists inform the system is on the blot. The software then captures one perfect image of all the different dyes, to ensure imaging fluorescent multiplex Westerns is quick and simple.
Laura Sullivan, Syngene’s Divisional Manager, explained: “Scientists want to use fluorescence to visualise proteins on Western blots because they can increase throughput by using the same blot to detect different proteins, something they can’t do using chemiluminescent-based blots. Additionally, it can often be difficult to accurately quantify proteins as some fluorescent dyes have overlapping spectra and membranes can auto-fluoresce, which interferes with detecting low abundance proteins. Using IR dyes can sometimes solve these problems.”
“Detecting IR dyes has proved difficult using CCD-based systems and so we are excited to have found filters and lighting combinations to allow our G:BOX Chemi to visualise multiplex Westerns of LI-COR IR dyes. This breakthrough means scientists with a G:BOX Chemi now have a sensitive, accurate method for imaging IR labelled proteins without having to buy an expensive laser-based scanner,” Laura Sullivan added.
Circle no. 180
Spotlight
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