Maximize your DNA

Numerous reports (1-8) show that exposure of DNA samples to UV radiation has a detrimental impact on downstream protocols. Transformation, transcription and PCR efficiencies are all reduced by 2-3 orders of magnitude when DNA samples are exposed to UV light - even for the brief period of time that it takes to excise a band from a gel. Also, the use of UV-exposed DNA templates can result in reduced fidelity and incorrect DNA replication. as well as impair the biological integrity of proteins encoded by the exposed DNA.

Described below are the results from experiments performed by various independent scientists showing the dramatic improvements (102 - 104 !) that can be achieved when DNA samples are viewed on a Dark Reader rather than a UV transilluminator.
1: Maximize Transformation

Method
In the experiments conducted by researchers at Epicentre, T7 DNA or wheat germ chromosomal DNA was incubated with End-Repair Enzyme Mix and the DNA fragments were resolved by electrophoresis and stained with SYBR Gold. During excision of the DNA bands from the gel, the DNA was exposed to either Dark Reader (DR) light or UV light (302 nm) for 30 seconds. The purified DNA was then ligated to the pWEB Cosmid Vector and the ligated DNA was packaged into lambda particles using the MaxPlax Lambda Packaging Extracts. EPI305 plating cells were then transfected, plated, and incubated overnight. Either plaques (for T7 DNA), or colonies (for wheat germ DNA) were counted.

maximum transformation using a Dark Reader

Results: Wheat Germ DNA
Exposure of the wheat germ DNA to 302 nm UV light for 30 seconds had a large impact on the cloning efficiency. The plating efficiency of the UV-exposed DNA was 2 x 104 cfu/ug DNA. In contrast, the plating efficiency of the Dark Reader (DR) - exposed DNA was 85-fold higher, with 1.7 x 106 cfu/ug DNA. These results demonstrate that UV-exposed DNA is significantly compromised in its ability to function well in such applications as the development of genomic libraries. For library construction an 85-fold reduction in cloning efficiency can be very significant, particularly for larger genomes.

Results: T7 Phage DNA
Viral infection with T7 phage requires that the cloned T7 DNA in each cosmid express the gene products, in active form, necessary for T7 phage production. DNA recovered from a gel visualized on a Dark Reader transilluminator produced a 220-fold greater number of plaques (2.2 x 106 pfu/ug DNA) than DNA recovered from a gel exposed to light from a UV transilluminator (1 x 104 pfu/ug DNA). These results are even more dramatic than those obtained with the wheat germ DNA. This clearly indicates that, in addition to affecting ligation and transformation efficiencies, the 30-second UV exposure compromised the activity and function of one or more of the gene products necessary for T7 phage production.
2: Maximize PCR
Dr. A. Michael Chin, the founder of Sequetech, tested a Dark Reader for visualizing DNA templates to be used in PCR reactions. Here are his notes:
Method
1. Ran some DNA fragments through an agarose gel in duplicate.
2. Cut the gel in half and stained one half with SYBR Gold and the other with ethidium bromide.
3. De-stained the ethidium bromide stained half. It was not necessary to de-stain the SYBR Gold half, saving some time.
4. Visualized the SYBR Gold half on the Dark Reader, took a picture and cut out the band I wanted.
5. Visualized the ethidium bromide half on a standard UV transilluminator, took a picture and cut out the band I wanted.
6. Purified both bands away from the agarose.
7. Performed PCR amplification of both purified DNA fragments. Started the amplifications with undiluted, 1/100 diluted and 1/10,000 diluted fragments for a total of 6 amplifications.
8. Ran all amplifications through an agarose gel and visualized them.

Results
All 3 of the Dark Reader amplifications maxed out the reactions while only the undiluted amplification of the UV-exposed template maxed out the reaction.

Conclusion
The DNA which was isolated using the Dark Reader was at least 10,000 times as amplifiable as the DNA isolated using ethidium bromide and a standard UV transilluminator. Since all of the Dark Reader amplifications were maxed out, they may be more that 10,000 times as amplifiable as the ethidium bromide/UV DNA.
3: Maximize Sequencing
In a second series of experiments to compare the effects of DR and UV exposure on down-stream DNA protocols, Epicentre scientists exposed DNA to either DR light (300 seconds) or 302 nm UV light (60 seconds) and then sequenced the DNA using SequiTherm EXCEL Long-Read DNA Sequencing kits. The results from a section of sequencing gel are shown below.
Notice the bands across all 4 lanes (BAFLs) after exposure to UV light for only 60 sec. However, after exposure on a Dark Reader transilluminator for 300 sec, DNA sequencing results are indistinguishable from those obtained with the zero-exposure control.
Optiimal DNA sequencing
1. Brunk, C.F. and L. Simpson. 1977. Comparison of various ultraviolet sources for fluorescent detection of ethidium bromide-DNA complexes in polyacrylamide gels. Anal. Biochem. 82:455-462.

2. Hartman, P. S. 1991. Transillumination can profoundly reduce transformation frequencies. BioTechniques 11:747-748.

3. Daum, H.A., H.G. White, C.M. Seidell and P.A. Johnson. 1991. Cloning, restriction digestion and DNA labeling of large DNA fragments in the presence of remelted SeaPlaque GTG agarose gels. BioTechniques 11:784-790.

4. Cariello, N. F., P. Keohavong, B.J.S. Sanderson and W.G. Thilly. 1988. DNA damage produced by ethidium bromide staining and exposure to ultraviolet light. Nuc. Acids Res. 16:4157.

5. Grundemann, D. and E. Schomig. 1996. Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. BioTechniques 21:898-903.

6. Paabo, S., D.M. Irwin and A.C. Wilson. 1990. DNA damage promotes jumping between templates during enzymatic amplification. J. Biol. Chem. 265:4718-4721.

7. Hoffman, L. 1996. T4 endonuclease V detects UV transilluminator damage to DNA in agarose gels. Epicentre Forum 3:4-5.Y

8. Jiang, C Ke, PA Mieczkowski, and PE Marszalek 2007 Detecting Ultraviolet Damage in Single DNA Molecules by Atomic Force Microscopy Biophysical Journal 93:1758-1767
Clare Chemical Research

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