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Results Addition of PCR enhancing agents PowerPoint Presentation
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Results Addition of PCR enhancing agents

Results Addition of PCR enhancing agents

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Results Addition of PCR enhancing agents

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  1. 4 SDS treatment [6] 5 Microwave oven [7] 6 FPFDc [8] Add 30 µL 2 g SDS/L to tube Add some cells to tube Add 100 µL EB to tubed Pick a colony and resuspend Heat in microwave for 1 mine Pick a colony and resuspend Vortex vigorously Add 25 µL PCR mix Heat at 95 °C for 15 min Spin for 1 min, use 1 µL Place on ice for 1 min Vortex 10 s Spin for 1 min, use 1 µL 7 Spheroplasts [9] 8 Freeze-thaw micowave oven [10] Add 10 µL enzyme incubation solution to tubef Add 5 µL of water to tube Pick a colony and resuspend Pick a colony and resuspend Incubate at 37 °C for 5 min Freeze for 10-15 min Use 0.5 µL for PCR Microwave for 1 min on high power Freeze for 10-15 min Add 20 µL PCR mix Comparison of colony PCR methods suitable for yeastSofie L. De Maeseneire, Wim K. Soetaert, Marjan De MeyDepartment of Biochemical and Microbial Technology, Ghent University, Coupure links 653, 9000 Ghent, Belgiumcontact: Sofie.DeMaeseneire@UGent.be, www.InBio.be Introduction Broad spectrum producing hosts and fundamental research models such as yeast can only be developed if one can rely both on sophisticated genetic 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.Yeast DNA to be used as a PCR template can be prepared by several quick methods involving heat treatment, freeze-thawing steps, alkali extraction, glass bead disruption and enzymatic digestion or the direct use of yeast cells in the PCR mixture. In this study several quick colony PCR methods were tested as to their ability to amplify plasmid (pDNA) or chromosomal DNA (gDNA) fragments of different length and to the reproducibility of the results. Tedious, costly or number-limiting methods and methods using toxic chemicals such as phenol or chloroform were excluded from this study. In a first step, addition of some compounds known to improve yield and consistency of PCR reactions was evaluated. Next, eight colony pre-treatment methods were compared. Methods Saccharomyces cerevisiae strain BY4742 was used for PCR on genomic DNA. S. cerevisiae strain Y12321 transformed with plasmid pSH47 was used for PCR on plasmid DNA. Results described for ‘fresh’ colonies refer to PCR’s performed on colonies incubated overnight, while results for ‘old’ colonies were obtained from colonies grown overnight and stored at 6°C for 3 to 4 days. The samples were used for 5 PCR reactions: 4 on genomic DNA (expecting 0.7, 0.9, 1.9 and 2.7 kb) and 2 on plasmid DNA (expecting 1.1 and 2.9 kb). The standard PCR-reaction mixture contained 1 unit Taq DNA polymerase (New England Biolabs), 1x Standard Taq Buffer, 0.2 mM dNTP mix, 0.4 µM primers and template. mQ-water was added up to 25 µL. All reactions were run in triplicate. In a first series of tests betain (1.25 M), Tween-20 (0.5 %, v/v), Triton X-100 (1 %, w/v) or gelatine (0,1 %, w/v) was added to the PCR mixtures, and cells were immediately added to the PCR mix. In a second series of tests cells were first pre-treated as described in Table 1. As recommended in the literature references, betain was added to the standard PCR mixtures of method 1 and 2, while Tween-20 or Triton X-100 was used in the mixtures of method 3 and 4. • Results • Addition of PCR enhancing agents • Adding cells immediately to the standard PCR-mix without enhancing agents resulted in consistent amplification of fragments • up to 1 kb from pDNA with fresh colonies (no reliable amplification of gDNA) • up to 1 kb from pDNA and 2 kb from gDNA using old colonies • For fresh colonies addition of betain to the standard PCR mix greatly enhanced results obtained with gDNA since fragments of 3 kb could be reliably amplified • For old colonies betain improved yield and specificity of the amplification • PCR’s were completely hampered by • adding gelatine • adding Tween-20 or Triton X-100 to the mixtures with fresh colonies • Colony pre-treatment methods • Best pre-treatment method for: • pDNA, fresh: 7 (spheroplast) or 8 (freeze-thawing @-20°C-microwave oven) • pDNA, old: 7 (spheroplast) or 8 (freeze-thawing @-20°C-microwave oven) • gDNA, fresh: 1 (boil in NaOH) or 8 (freeze-thawing @-20°C-microwave oven) • gDNA, old: 5 (microwave oven) • Longest fragment consistently obtained: • pDNA, fresh: 1 kb • pDNA, old: 1 kb • gDNA, fresh: 2 kb • gDNA, old: 3 kb Table 1. Colony pre-treatment methods 1 Boil in NaOH [3] 2 Boil in cracking buffer [4] 3 Freeze-thaw SDS [5] Add 10 µL of 0.02 M NaOH to tube Add 40 µL of to tube Add 50 µL lysis buffer to tubea Pick a colony and resuspend Pick a colony and resuspend Pick a colony and resuspend Incubate at 95 °C for 10 min Add 10 μL cracking bufferb Place at -80 °C for 2 min Spin for 1 min, use 1 µL Pipette mix Transfer to 95 °C for 1 min Incubate at 95 °C for 10 min Repeat freeze-thaw 2 times Spin for 1 min, use 1 µL Vortex 30 s Spin for 1 min, use 1 µL a Lysis buffer: 2 % Triton X-100 (v/v), 2 g SDS/L, 100 mM NaCl, 10 mM TrisCl pH 8.0, 1 mM EDTA b Cracking buffer: 0.5 M NaOH, 5 g SDS/L, 500 g sucrose/L c FPFD: Fast Preparation of Fungal DNA [8] d Extraction buffer: 5 mM carbonate buffer pH 9.6, 2 % PVP 40 (w/v), 0.2 % BSA, 0.05% Tween 20 e Together with a beaker of water f Enzyme incubation solution: 1.2 M sorbitol, 100 mM Na-phosphate pH 7.4, 2.5 g Zymolase/L (ICN) • Conclusions • The colony PCR method to be used depends both on the nature of the colony (fresh or old) and the nature of the DNA (plasmid or chromosomal) • Adding betain to the PCR mixture enhances results both with fresh colonies (fragment length gDNA) and with old colonies (yield and specificity) • The microwave oven pre-treatment method is the most efficient for gDNA amplification (up to 3 kb) with old colonies • A PCR mix with betain and cells added straight to the mix is the fastest, most reliable and performant for pDNA (fresh/old, to 1kb) and gDNA (fresh, to 3kb) • Storing plates with colonies for a few days at -6 °C significantly influences PCR results References [1] Roberts, I.N. Biotechnology Letters, 2011. 33(3): p. 477-487. [6] Akada, R. Biotechniques, 2000. 28(4): p. 668-+. [2] Shibasaki, S. Journal of the Pharmaceutical Society of Japan, 2010. 130(11): p. 1437-1444. [7] Lisby, M. available from: http://www1.bio.ku.dk/english/research/fg/transkription/resources/protocols. [3] Wang, H. Analytical Biochemistry, 1996. 237(1): p. 145-146. [8] Liu, K.H. Journal of Microbiological Methods, 2011. 85(2): p. 170-172. [4] Personal communication Ajikumar P. K. Parayil, Bioinformatics and Metabolic Engineering, MIT, Cambridge [9] Ling, M.F. Nucleic Acids Research, 1995. 23(23): p. 4924-4925. [5] Harju, S. Bmc Biotechnology, 2004. 4. [10] The Murray Lab. available from: http://www.mcb.harvard.edu/murray/colony_pcr.html.