Replacing UV with Blue Light during DNA Purification Increases the Efficiency of Ligation-Independent Cloning
Trends in Peptide and Protein Sciences,
Vol. 3 (2018),
1 June 2018
,
Page 1-8 (e1)
https://doi.org/10.22037/tpps.v3i0.19997
Abstract
Ligation-independent cloning is a simple method that provides several advantages over conventional cloning. However, the efficiency of ligation-independent cloning is considerably lower than that of conventional methods. Several studies have shown that competent cells used for ligation-independent cloning should preferably have a transformation efficiency of 106-107 cfu/μg DNA. Although such levels can be easily achieved using standard protocols with most Escherichia coli strains, some strains attain mush lower values. When such strains have to be used for ligation-independent cloning, certain measures need to be taken to avoid any situation that may further decrease the efficiency of the process. These measures, however, are usually time-consuming. This problem is exacerbated by the fact that some strains such as BL21 (DE3) appear to be intrinsically unsuited for ligation-independent cloning. Here we suggest that by avoiding DNA damage during purification simply by replacing UV transilluminators with blue light systems BL21 (DE3) cells with a transformation efficiency of 105 cfu/μg DNA can satisfactorily be used for ligation-independent cloning without any additional steps.
HIGHLIGHTS
•Using blue light instead of UV light increases the efficiency of LIC.
•A simple LED blue light projector combined with a filter can be used for this purpose.
•This approach is not recommended for applications that require higher sensitivity.
- Blue light
- DNA damage
- Ligation-independent cloning
- Ultraviolet
How to Cite
References
Aslanidis, C. and P.J. De Jong, (1990).”Ligation-independent cloning of PCR products (LIC-PCR).” Nucleic Acids Research, 18(20): 6069-6074.
Beyer, H.M., Gonschorek, P., Samodelov, S.L., Meier, M., Weber, W. and M.D. Zurbriggen, (2015).”AQUA cloning: A versatile and simple enzyme-free cloning approach.” PLOS ONE, 10(9): e0137652.
Cadet, J. and J.R. Wagner, (2013).”DNA base damage by reactive oxygen species, oxidizing agents, and UV radiation.” Cold Spring Harbor Perspectives in Biology, 5(2): a012559.
Chan, W.T., Verma, C.S., Lane, D.P. and S.K.E. Gan, (2013).”A comparison and optimization of methods and factors affecting the transformation of Escherichia coli.” Bioscience Reports, 33(6): e00086.
Dutra, B.E., Sutera, V.A.Jr. and S.T. Lovett, (2007).”RecAindependent recombination is efficient but limited by exonucleases.” Proceedings of the National Academy of Sciences of the United States of America, 104(1): 216-221.
Godley, B.F., Shamsi, F.A., Liang, F.Q., Jarrett, S.G., Davies, S. and M. Boulton, (2005).”Blue light induces mitochondrial DNA damage and free radical production in epithelial cells.” The Journal of Biological Chemistry, 280(22): 21061-21066.
Grundemann, D. and E. Schomig, (1996).”Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light.” Biotechniques, 21(5): 898-903.
Jacobus, A.P. and J. Gross, (2015).”Optimal cloning of PCR fragments by homologous recombination in Escherichia coli.” PLOS ONE, 10(3): e0119221.
Klock, H.E., Koesema, E.J., Knuth, M.W. and S.A. Lesley, (2008).”Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts.” Proteins, 71(2): 982-994.
Kostylev, M., Otwell, A.E., Richardson, R.E. and Y. Suzuki, (2015).”Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies.” PLOS ONE, 10(9): e0137466.
Kuzminov, A., Schabtach, E. and F.W. Stahl, (1994).”Chi sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease.” The EMBO Journal, 13(12): 2764-2776.
Li, C., Wen, A., Shen, B., Lu, J., Huang, Y. and Y. Chang, (2011).”FastCloning: A highly simplified, purification-free, sequenceand ligation-independent PCR cloning method.” BMC Biotechnology, 11(1): 92.
Robin, G. and P. Martineau, (2012).”Synthetic customized scFv libraries.” Methods in Molecular Biology, 907: 109-122.
Sambrook, J. and D.W. Russell, (2001). “Molecular Cloning: A Laboratory Manual.” 3rd ed., Cold Spring Harbor Laboratory Press, New York, pp. 5.1-5.90.
Seidman, C.E., Struhl, K., Sheen, J. and T. Jessen, (2001).”Introduction of plasmid DNA into cells.” Current Protocols in Molecular Biology, 37: 1.8.1-1.8.10.
Sharafi, Z., Barati, M., Khoshayand, M.R. and S. Adrangi, (2017). “Screening for type II L-asparaginases: Lessons from the genus Halomonas.” Iranian Journal of Pharmaceutical Research, 16(4): 1565- 1573.
Stevenson, J., Krycer, J.R., Phan, L. and A.J. Brown, (2013).”A practical comparison of ligation-independent cloning techniques.” PLOS ONE, 8(12): e83888.
Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S. and T.L. Madden, (2012).”Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction.” BMC Bioinformatics, 13: 134-134.
You, C., Zhang, X.Z. and Y.H.P. Zhang, (2012).”Simple Cloning: Direct transformation of PCR product (DNA multimer) to Escherichia coli and Bacillus subtilis.” Applied and Environmental Microbiology, 78(5): 1593-1595.
- Abstract Viewed: 477 times
- PDF Downloaded: 235 times