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, widulle@vilber.de
Keywords: transilluminator, multiband, UV, gel documentation, image acquisition, cloning efficiency
Stichworte: UV-Tisch, Multiband, UV, Geldokumentation, Bildaufnahme, Klonierungseffizienz
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 ampi-
cillin 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).
Number of white colonies in %
of the total number of colonies
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.
Cloning efficiency
50,00%
Standard transilluminator, UV
B
Super-Bright
transilluminator, UV B, 70%
light intensity
selected
40,00%
30,00%
20,00%
10,00%
0,00%
20
80
140
200
Time of UV irradiation (sec)
260
Fig. 1a: Cloning efficiency of chloramphenicol acetyl transferase (cat) in
%. The number of white colonies is reduced already after short exposure
to UV B light.
Vilber Lourmat Deutschland GmbH, Wielandstrasse 2, D-88436 Eberhardzell
Tel: +49 (0)7355-931380, Fax: +49 (0)7355-931379, www.vilber.de
Application Note VL0801
Expression of functional cat
Standard transilluminator, UV
B
Super-Bright
transilluminator, UV B, 70%
light intensity
selected
80,00%
60,00%
40,00%
20,00%
0,00%
20
80
140
Time of UV irradiation (sec)
200
Fig. 1b: Analysis for expression of chloramphenicol acetyl transferase (cat).
The number of chloramphenicol-resistant colonies is reduced already after
short exposure to UV B light.
Number of white colonies in %
of the total number of colonies
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).
Cloning efficiency
40,00%
35,00%
30,00%
25,00%
20,00%
15,00%
10,00%
5,00%
0,00%
Multiband
transilluminator, UV B selected
Multiband
transilluminator, UV A selected
20
75
135
Time of UV irradiation (sec)
255
Fig. 2a: Cloning efficiency of chloramphenicol acetyl transferase (cat) in
%. The number of white colonies remains more stable when the DNA fragment is exposed to UV A light.
Number of resistant colonies
in % of the white colonies
Number of resistant colonies
in % of the white colonies
Expression of functional cat
100,00%
100,00%
Multiband
transilluminator, UV B selected
Multiband
transilluminator, UV A selected
80,00%
60,00%
40,00%
20,00%
0,00%
20
75
135
Time of UV irradiation (sec)
255
Fig. 2b: Analysis for expression of chloramphenicol acetyl transferase (cat).
The number of chloramphenicol resistant colonies remains more stable
when the DNA fragment is exposed to UV A light.
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 SYBRTM to Q-DotTM.
Vilber Lourmat Deutschland GmbH, Wielandstrasse 2, D-88436 Eberhardzell
Tel: +49 (0)7355-931380, Fax: +49 (0)7355-931379, www.vilber.de