An application note to show which UV wavelength is the best for cloning experiments.

(Armin Günther and Reinhold Horlacher, Trenzyme GmbH, Konstanz, Germany, www.trenzyme.com; Karin Widulle, Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany, info@vilber.de)

Introduction

UV transilluminators are most commonly used in modern laboratories to visualize nucleic acids stained with ethidium bromide or other dyes. There are two different main applications:

- Analytical gel documentation, i.e. acquisition of images for printing/storage on files

- Preparative work, i.e. excision of DNA bands from gels, e.g. for cloning purposes

These two applications require transilluminators with different characteristics. Short wave or medium wave UV light (UV C and B) is crucial for analytical gel documentation to enhance the image brightness and to reach a maximum of contrast. On the other side, long wave UV light (UV A) is recommended
for preparative work, as short and medium wave UV light can cause severe damages to the DNA. During UV irradiation, two adjacent thymine bases can be covalently crosslinked so that thymine-dimers are formed. These can cause mutations or the replication may be interfered.

What does that mean for the laboratory work? Is it necessary to possess two distinct transilluminators, one for analytical, the other for preparative applications? What about Multiband transilluminators with possibility to select between different wavelengths?

In order to get more insight into this topic, the
following experiments were designed and performed in collaboration with Trenzyme GmbH, experts in cloning of DNA.

Method

Identical aliquots of a PCR-based DNA fragment (chloramphenicol acetyl transferase (cat) from pACYC184) were separated 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 supplemented with 200 µg/ml ampicillin and 40 µg/ml X-Gal. White colonies indicated the presence of inserts. Aliquots of the white colonies were selected and transferred to LB medium supplemented with 30 µg/ml chloramphenicol and ampicillin to analyse for expression of functional cat. As the UV light can cause extensive crosslinking between nucleotides, resistance to chloramphenicol may be abolished.

The following UV transilluminators were used for comparison:

- Standard transilluminator, UV B

- Super-Bright transilluminator, UV B, 70% light intensity selected

- Multiband transilluminator, UV B selected

- Multiband transilluminator, UV A selected

Results

Fig. 1 shows the efficiency of cloning (Fig. 1a) and expression of functional cat gene (Fig. 1b) after exposure to the Standard transilluminator and to the Super-Bright transilluminator with 70% light intensity selected. Already after exposure times of 80 sec at UV B light, the number of white colonies, i.e.the insert rate, was drastically reduced. This applied to both transilluminators (Fig. 1a). In parallel, the number of chloramphenicol-resistant colonies decreased with the exposure time. After 2 min incubation with UV B light, more than 50% of the white colonies originally selected did not carry an intact, functional insert (Fig. 1b).

Fig. 2 shows the efficiency of cloning (Fig. 2a) and expression of functional cat gene (Fig. 2b) after exposure to the Multiband transilluminator with UV B light selected and to the Multiband transilluminator with UV A light selected. When excising the DNA fragments at UV B light, the number of white colonies was clearly reduced after 75 sec (Fig. 2a), as well as the number of chloramphenicol-resistant colonies (Fig. 2b). When excising the DNA fragments at UV A light, the number of white colonies remained much more stable even at incubation times of more than 2 min (Fig. 2a). The number of chloramphenicol-resistant colonies remained very high, indicating the cloning of intact DNA without UV induced mutations (Fig. 2b).

Conclusions

The experiments showed that the wavelength of the UV light used during excision of DNA fragments from agarose gels is crucial for the quality of the DNA. Exposure to UV B light for more than one minute drastically reduced the cloning efficiency as well as the number of chloramphenicol-resistant
colonies, indicating that mutations may have occured which abolish the function of the cat gene. UV A light instead seems to have almost no negative influence on the integrity of the nucleic acid to be cloned, even after exposure times of more than two minutes.

The following conclusions can be drawn:
For preparative DNA work, UV A transilluminators are superior to UV B transilluminators. This applies especially if high numbers of samples have to be excised from agarose gels, as there will be a long exposure to UV light. Multiband transilluminators 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 transilluminator ECP-26.LMX in addition to the existing product range of Multiband and Super-Bright transillluminators.
This new transilluminator will set the benchmark in performance and versatility. It offers all the described advantages of Multiband transilluminators for preparative work. In addition, 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 SYBR^TM to Q-Dot^TM.