Even a 30-second exposure to ethidium bromide and UV reduces cloning efficiency. Gels loaded with equal amounts of a PCR product (1.25 kb gene fragment from Ultimate™ ORF IOH# 11050) were stained with either SYBR® Safe DNA gel stain (1:10,000 in TBE) or ethidium bromide (0.5 µg/ml in TBE) following electrophoresis. The gel stained with SYBR® Safe stain was visualized on a blue-light box with light emission identical to that produced by the Safe Imager™ transilluminator (S37102). The ethidium bromide-stained gel was visualized using UV transillumination. Bands were excised at defined exposure times. DNA was purified from the gel fragments under identical conditions and used in parallel sub-cloning reactions.
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Following transformation into OneShot® TOP 10 chemically competent bacteria, three serial dilutions were plated and colonies counted using an Alpha Innotech imaging system. A plot of the experiment is shown here.Figure 2. Use of SYBR® Safe Stain and the Safe Imager™ Transilluminator causes minimal DNA damage. Equivalent fractions of supercoiled DNA stained with SYBR® Safe DNA Gel Stain or ethidium bromide were exposed to blue light (Safe Imager™ Transilluminator, Invitrogen) (A) or UV light (B), respectively, for defined periods of time and evaluated by agarose electrophoresis.
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A slowermigrating species is indicative of a linear or relaxed circular vector resulting from DNA nicking or strand breaks.