Vilber.de

Application Note VL0801
Which UV transilluminator should be used for preparative DNA work?
Armin Günther and Reinhold Horlacher, Trenzyme GmbH, Konstanz, Germany, www.trenzyme.com Karin Widulle, Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany, [email protected] Keywords: transilluminator, multiband, UV, gel documentation, image acquisition, cloning efficiencyStichworte: UV-Tisch, Multiband, UV, Geldokumentation, Bildaufnahme, Klonierungseffizienz Introduction:
cillin to analyse for expression of functional cat. As the UV UV transilluminators are most commonly used in modern light can cause extensive crosslinking between nucleotides, laboratories to visualize nucleic acids stained with ethidium resistance to chloramphenicol may be abolished. bromide or other dyes. There are two different main applica- The following UV transilluminators were used for compari-tions: • Analytical gel documentation, i.e. acquisition of images • Super-Bright transilluminator, UV B, 70% light intensity • Preparative work, i.e. excision of DNA bands from gels, • Multiband transilluminator, UV B selected These two applications require transilluminators with diffe- • Multiband transilluminator, UV A selected
rent characteristics. Short wave or medium wave UV light (UV
C and B) is crucial for analytical gel documentation to enhan-
ce the image brightness and to reach a maximum of contrast.
On the other side, long wave UV light (UV A) is recommended Results:
for preparative work, as short and medium wave UV light can Fig. 1 shows the efficiency of cloning (Fig. 1a) and expression cause severe damages to the DNA. During UV irradiation, two of functional cat gene (Fig. 1b) after exposure to the Stan- adjacent thymine bases can be covalently crosslinked so that dard transilluminator and to the Super-Bright transillumina- thymine-dimers are formed. These can cause mutations or tor with 70% light intensity selected. Already after exposure times of 80 sec at UV B light, the number of white colonies, i.e. What does that mean for the laboratory work? Is it necessary the insert rate, was drastically reduced. This applied to both to possess two distinct transilluminators, one for analytical, transilluminators (Fig. 1a). In parallel, the number of chloram- the other for preparative applications? What about Multiband phenicol-resistant colonies decreased with the exposure time. transilluminators with possibility to select between different After 2 min incubation with UV B light, more than 50% of wavelengths? In order to get more insight into this topic, the the white colonies originally selected did not carry an intact, following experiments were designed and performed in colla- functional insert (Fig. 1b). boration with Trenzyme GmbH, experts in cloning of DNA.
Cloning efficiency
Identical aliquots of a PCR-based DNA fragment (chloram- phenicol acetyl transferase (cat) from pACYC184) were sepa- rated on 1% agarose gels and excised after defined exposure times ranging from 20 sec up to 260 sec. Using Trenzyme´s Alligator cloning kit, the fragments were cloned into pAlli10, transformed into E.coli TZ101 alpha and plated on LB medium Number of white colonies in %
supplemented with 200 µg/ml ampicillin and 40 µg/ml X-Gal. of the total number of colonies
Time of UV irradiation (sec)
White colonies indicated the presence of inserts. Aliquots of Fig. 1a: Cloning efficiency of chloramphenicol acetyl transferase (cat) in the white colonies were selected and transferred to LB medi- %. The number of white colonies is reduced already after short exposure um supplemented with 30 µg/ml chloramphenicol and ampi- to UV B light.
Vilber Lourmat Deutschland GmbH, Wielandstrasse 2, D-88436 EberhardzellTel: +49 (0)7355-931380, Fax: +49 (0)7355-931379, www.vilber.de Application Note VL0801
Expression of functional cat
Expression of functional cat
in % of the white colonies
in % of the white colonies
Number of resistant colonies
Number of resistant colonies
Time of UV irradiation (sec)
Time of UV irradiation (sec)
Fig. 1b: Analysis for expression of chloramphenicol acetyl transferase (cat). Fig. 2b: Analysis for expression of chloramphenicol acetyl transferase (cat). The number of chloramphenicol-resistant colonies is reduced already after The number of chloramphenicol resistant colonies remains more stable when the DNA fragment is exposed to UV A light.
Conclusions:
Fig. 2 shows the efficiency of cloning (Fig. 2a) and expression The experiments showed that the wavelength of the UV light of functional cat gene (Fig. 2b) after exposure to the Multi- used during excision of DNA fragments from agarose gels is band transilluminator with UV B light selected and to the crucial for the quality of the DNA. Exposure to UV B light for Multiband transilluminator with UV A light selected. When more than one minute drastically reduced the cloning effi-excising the DNA fragments at UV B light, the number of ciency as well as the number of chloramphenicol-resistant white colonies was clearly reduced after 75 sec (Fig. 2a), as colonies, indicating that mutations may have occured which well as the number of chloramphenicol-resistant colonies abolish the function of the cat gene. UV A light instead seems (Fig. 2b). When excising the DNA fragments at UV A light, the to have almost no negative influence on the integrity of the number of white colonies remained much more stable even nucleic acid to be cloned, even after exposure times of more at incubation times of more than 2 min (Fig. 2a). The number than two minutes. of chloramphenicol-resistant colonies remained very high, indicating the cloning of intact DNA without UV induced mu- The following conclusions can be drawn:tations (Fig. 2b).
For preparative DNA work, UV A transilluminators are superior to UV B transilluminators. This applies especially if high num-bers of samples have to be excised from agarose gels, as there Cloning efficiency
will be a long exposure to UV light. Multiband transillumina- tors with wavelength selector are a good choice, allowing to do preparative work and analytical imaging with one system. As a consequence of the presented findings, Vilber Lourmat has developed the Super-Bright-Multiband transillumina- tor ECP-26.LMX in addition to the existing product range of Multiband and Super-Bright transillluminators. Number of white colonies in % of the total number of colonies
This new transilluminator will set the benchmark in perfor- Time of UV irradiation (sec)
mance and versatility. It offers all the described advantages Fig. 2a: Cloning efficiency of chloramphenicol acetyl transferase (cat) in of Multiband transilluminators for preparative work. In ad- %. The number of white colonies remains more stable when the DNA frag- dition, the Super-Bright technology guarantees results with the optimum signal quality for analytical applications, as it is required for digital imaging. A broad range of fluorescent dyes can be used, from SYBRTM to Q-DotTM.
Vilber Lourmat Deutschland GmbH, Wielandstrasse 2, D-88436 EberhardzellTel: +49 (0)7355-931380, Fax: +49 (0)7355-931379, www.vilber.de

Source: http://www.vilber.de/fileadmin/user_upload/Downloads/Application_Note_VL0801.pdf