School of Engineering and Science - Jacobs University

More documents

Recommendations

Info

Material and Methods extraction. For this, 1 volume of chloroform/isoamyl alcohol (24:1; v/v) was added to the sample, mixed and centrifuged for 10 min. in a microcentrifuge. Subsequently, the upper phase was transferred to a fresh plastic reaction tube and mixed with 1 volume of phenol/choroform/isoamyl alcohol (25:24:1; v/v). After centrifugation, the upper phase was transferred to a fresh plastic reaction tube, and mixed with 1 volume of chloroform/isoamyl alcohol (24:1; v/v) to remove residues of phenol. After centrifugation, the upper phase was transferred to a fresh plastic reaction tube and the genomic DNA was precipitated by addition of 0.6 volumes of isopropanol. The precipitate was harvested by 30 min centrifugation in a microcentrifuge and washed once with 70 % ethanol. The resulting DNA-pellet was dried and resuspended in 50- 300 µl of sterile water. DNA was stored at –20°C. 4.2.3 DNA separation by gel electrophoresis DNA-fragments can be separated in an agarose gel matrix upon application of an electric field. Migration of the DNA fragments depends on the electrical charge of the fragment and its structural interactions with the polymer mesh of the agarose gel. Therefore for linear DNA fragments the migration distance correlates to the fragment size. Upon addition of ethidium bromide which is intercalated into GC-pairs, DNA becomes fluorescent and can be visualized under UV-light (Sambrook et al., 1989). Agarose gels were prepared in TAE buffer (1x: 40 mM Tris-acetate, 1.3 mM EDTA-Na, 0.47 mM acetic acid). For agarose gels that were subsequently blotted to a membrane an agarose concentration of 0.8 % was used, while gels for standard applications contained 1 % agarose. DNA samples were mixed with 1/6 volume of loading buffer (6x: 0.25 % bromphenol blue, 0.25 % xylene cyanol, 40 % sucrose) and separated at 80 V in TAE buffer. The size of DNA fragments was determined by parallel loading to the gel of the 1-kb DNA ladder (GibcoBRL) or the gene ruler (MBI fermentas). Both markers are suitable to estimate the size of DNA fragments in a range from 500 bp to 12 kb. After electrophoresis, the gel was 31
Material and Methods stained in ethidium bromide solution (10 µg/ml in TAE buffer) for 5-15 min. Then the gel was analysed and documented under UV-light using the gel jet imager system (INTAS). 4.2.4 Polymerase chain reaction (PCR) Polymerase chain reaction (PCR) is a method used to amplify a specific DNA sequence in vitro by repeated cycles of synthesis with specific primers and thermostable Taq DNA polymerase (Saiki et al., 1988). Specific primers are complementary to sequences that lie on opposite strands of the template DNA and flank the segment of DNA that is to be amplified. The template DNA was first denatured by heating in the presence of a large molar excess of each of the two primers and dNTPs. The reaction mixture was then cooled to a temperature that allows the primers to anneal to their target sequences, and subsequently the annealed primers were extended with DNA polymerase. Cycles of denaturation, annealing, and DNA synthesis were repeated several times. Reaction mixture was prepared according to the supplier’s manual. As the primers used in this study were designed from heterologues sequence information, touch-down PCR was applied to enhance the yield of product. A typical PCR thermoprofile was programmed as follows: Initial denaturation 95°C 5 min 1x Denaturation 95°C 1 min Hybridisation 65-50° 30 sec. 10x Polymerisation 65°C 1 min/kb Denaturation 95°C 1 min Hybridisation 55°C-45°C (-0.5°C/cycle) 30 sec. 25x Polymerisation 65°C 1 min/kb Final extension 65°C 5 min 32
Page 1 and 2: Siderophore production of Pseudomon
Page 3 and 4: Acknowledgements This project was s
Page 5 and 6: Abbreviations Ach achromobactin-lik
Page 7 and 8: Contents 4.1.2 Pseudomonas syringae
Page 9 and 10: Contents 5.3 Influence of sideropho
Page 11 and 12: Introduction 2 Introduction Like hu
Page 13 and 14: Introduction In contrast to the rhi
Page 15 and 16: Introduction 2.1.2 Host, pathogen a
Page 17 and 18: Introduction The relationship betwe
Page 19 and 20: Introduction developement of resist
Page 21 and 22: Introduction Fe 2+ + H 2 O 2 → Fe
Page 23 and 24: Introduction 1997a; May et al., 199
Page 25 and 26: Introduction Pss22d demonstrated a
Page 27 and 28: Introduction 2.4 Aim of this study
Page 29 and 30: Material and Methods Tab. 4. Antibi
Page 31 and 32: Material and Methods Iron-free 5b m
Page 33 and 34: Material and Methods Solution3, car
Page 35 and 36: Material and Methods 3.6 Plasmids T
Page 37 and 38: Material and Methods PCR-Screening
Page 39: Material and Methods on ice for 10
Page 43 and 44: Material and Methods 4.2.5.3 Conver
Page 45 and 46: Material and Methods 4.2.5.9 Ligati
Page 47 and 48: Material and Methods strains were s
Page 49 and 50: Material and Methods 9.5). For sign
Page 51 and 52: Material and Methods gentle shaking
Page 53 and 54: Material and Methods columns were w
Page 55 and 56: Results 5 Results 5.1 Analysis of s
Page 57 and 58: Results The 13-kb open reading fram
Page 59 and 60: Results If integration is due to a
Page 61 and 62: Results Yersiniabactin is a siderop
Page 63 and 64: Results Next, a large number of tra
Page 65 and 66: Results Nevertheless the site of tr
Page 67 and 68: Results Achr2_KpnI_rev binding site
Page 69 and 70: Results Interestingly the mutant Ps
Page 71 and 72: Results XAD4-purification yielded i
Page 73 and 74: Results After changing the mobile p
Page 75 and 76: Results P P A Fig. 21. Isoelectic f
Page 77 and 78: Results reduced in comparison to th
Page 79 and 80: Results A-E represents single inocu
Page 81 and 82: Results Tab. 10. Development of pop
Page 83 and 84: Results Tab. 11. Distribution of th
Page 85 and 86: Discussion bacteria with redundande
Page 87 and 88: Discussion 6.2 Possible regulation
Page 89 and 90: Discussion stationary phase transit
Page 91 and 92:
Discussion and bacterium (Loper et
Page 93 and 94:
Literature Bultreys, A., Gheysen, I
Page 95 and 96:
Literature Galperin, M. Y. (2006).
Page 97 and 98:
Literature Mazzola, M. (2002). Mech
Page 99 and 100:
Literature Schubert, S., Rakin, A.,
Page 101:
Literature Zou, J., Rodriguez-Zas,
show all

School of Engineering and Science - Jacobs University

Create successful ePaper yourself

Delete template?

Save as template?