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Comparison of colony PCR methods for yeast

Sofie De Maeseneire UGent, Wim Soetaert UGent and Marjan De Mey UGent (2012) Applied Synthetic Biology in Europe, 1st Symposium, Abstracts. p.62-62
abstract
Broad spectrum producing hosts and fundamental research models such as yeast can only be developed if one can rely both on sophisticated engineering techniques, as for example required in the emerging fields of synthetic biology and combinatorial engineering, and consistent basic molecular tools [1]. One of these basic tools needed for any type of genetic engineering is a quick and reliable method for screening transformants. In this study nine colony PCR methods were tested for the amplification of plasmid or chromosomal DNA from fresh and older colonies of Saccharomyces cerevisiae. The methods involved boiling in NaOH [2] or in cracking buffer, freeze-thawing in lysis buffer [3] or in the microwave oven [4], SDS treatment [5], heating in the microwave oven [6], spheroplast preparation [7], fast preparation of fungal DNA described by Liu et al. [8], and the use of untreated yeast cells in the PCR. For fresh colonies, freeze-thawing cycles (-20 °C/microwave oven) gave the most relable results, obtaining fragments up to 1 kb and 2 kb from plasmid and chromosomal DNA, respectively. With older cells, amplification of 3 kb genomic DNA fragments and 1 kb plasmid DNA fragments was consistently achieved by heating cells in the microwave oven. As for the addition PCR enhancing agents, it can be concluded that betain or triton are more beneficial than tween. 1. Roberts, I.N. and S.G. Oliver. Biotechnology Letters, 2011. 33(3): p. 477-487. 2. Wang, H., S.E. Kohalmi, and A.J. Cutler. Analytical Biochemistry, 1996. 237(1): p. 145-146. 3. Harju, S., H. Fedosyuk, and K.R. Peterson. Bmc Biotechnology, 2004. 4. 4. The Murray Lab from: http://www.mcb.harvard.edu/murray/colony_pcr.html. 5. Akada, R., T. Murakane, and Y. Nishizawa. Biotechniques, 2000. 28(4): p. 668-+. 6. Lisby, M. from: www1.bio.ku.dk/english/research/fg/transkription/resources/protocols/colonypcr/. 7. Ling, M.F., F. Merante, and B.H. Robinson. Nucleic Acids Research, 1995. 23(23): p. 4924-4925. 8. Liu, K.H., Y.L. Yeh, and W.C. Shen. Journal of Microbiological Methods, 2011. 85(2): p. 170-172.
Please use this url to cite or link to this publication:
author
organization
year
type
conference
publication status
published
subject
keyword
Yeast PCR
in
Applied Synthetic Biology in Europe, 1st Symposium, Abstracts
pages
62 - 62
publisher
European Federation of Biotechnology (EFB)
conference name
1st Symposium on Applied Synthetic Biology in Europe
conference location
Barcelona, Spain
conference start
2012-02-06
conference end
2012-02-08
language
English
UGent publication?
yes
classification
C3
id
2081498
handle
http://hdl.handle.net/1854/LU-2081498
date created
2012-04-05 14:08:13
date last changed
2015-03-13 15:18:01
@inproceedings{2081498,
  abstract     = {Broad spectrum producing hosts and fundamental research models such as yeast  can only be developed if one can rely both on sophisticated engineering techniques, as for example required in the emerging fields of synthetic biology and combinatorial engineering, and consistent basic molecular tools [1]. One of these basic tools needed for any type of genetic engineering is a quick and reliable method for screening transformants. 
In this study nine colony PCR methods were tested for the amplification of plasmid or chromosomal DNA from fresh and older colonies of Saccharomyces cerevisiae. The methods involved boiling in NaOH [2] or in cracking buffer, freeze-thawing in lysis buffer [3] or in the microwave oven [4], SDS treatment [5], heating in the microwave oven [6], spheroplast preparation [7], fast preparation of fungal DNA described by Liu et al. [8], and the use of untreated yeast cells in the PCR. For fresh colonies, freeze-thawing cycles (-20 {\textdegree}C/microwave oven) gave the most relable results, obtaining fragments up to 1 kb and 2 kb from plasmid and chromosomal DNA, respectively. With older cells, amplification of 3 kb genomic DNA fragments and 1 kb plasmid DNA fragments was consistently achieved by heating cells in the microwave oven. As for the addition PCR enhancing agents, it can be concluded that betain or triton are more beneficial than tween.
1.\unmatched{0009}Roberts, I.N. and S.G. Oliver. Biotechnology Letters, 2011. 33(3): p. 477-487.
2.\unmatched{0009}Wang, H., S.E. Kohalmi, and A.J. Cutler. Analytical Biochemistry, 1996. 237(1): p. 145-146.
3.\unmatched{0009}Harju, S., H. Fedosyuk, and K.R. Peterson. Bmc Biotechnology, 2004. 4.
4.\unmatched{0009}The Murray Lab from: http://www.mcb.harvard.edu/murray/colony\_pcr.html.
5.\unmatched{0009}Akada, R., T. Murakane, and Y. Nishizawa. Biotechniques, 2000. 28(4): p. 668-+.
6.\unmatched{0009}Lisby, M. from: www1.bio.ku.dk/english/research/fg/transkription/resources/protocols/colonypcr/.
7.\unmatched{0009}Ling, M.F., F. Merante, and B.H. Robinson. Nucleic Acids Research, 1995. 23(23): p. 4924-4925.
8.\unmatched{0009}Liu, K.H., Y.L. Yeh, and W.C. Shen. Journal of Microbiological Methods, 2011. 85(2): p. 170-172.},
  author       = {De Maeseneire, Sofie and Soetaert, Wim and De Mey, Marjan},
  booktitle    = {Applied Synthetic Biology in Europe, 1st Symposium, Abstracts},
  keyword      = {Yeast PCR},
  language     = {eng},
  location     = {Barcelona, Spain},
  pages        = {62--62},
  publisher    = {European Federation of Biotechnology (EFB)},
  title        = {Comparison of colony PCR methods for yeast},
  year         = {2012},
}

Chicago
De Maeseneire, Sofie, Wim Soetaert, and Marjan De Mey. 2012. “Comparison of Colony PCR Methods for Yeast.” In Applied Synthetic Biology in Europe, 1st Symposium, Abstracts, 62–62. European Federation of Biotechnology (EFB).
APA
De Maeseneire, S., Soetaert, W., & De Mey, M. (2012). Comparison of colony PCR methods for yeast. Applied Synthetic Biology in Europe, 1st Symposium, Abstracts (pp. 62–62). Presented at the 1st Symposium on Applied Synthetic Biology in Europe, European Federation of Biotechnology (EFB).
Vancouver
1.
De Maeseneire S, Soetaert W, De Mey M. Comparison of colony PCR methods for yeast. Applied Synthetic Biology in Europe, 1st Symposium, Abstracts. European Federation of Biotechnology (EFB); 2012. p. 62–62.
MLA
De Maeseneire, Sofie, Wim Soetaert, and Marjan De Mey. “Comparison of Colony PCR Methods for Yeast.” Applied Synthetic Biology in Europe, 1st Symposium, Abstracts. European Federation of Biotechnology (EFB), 2012. 62–62. Print.