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| - | == Transformation of Bacteria ==
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| - | For enrichment of vectors, E .coli DH5α were used. For the transformation 100 µl of the competent cells were thawed on ice and 50 – 400 ng DNA solution added (depending on the concentration of the DNA solution). After a 30-60 minute incubation on ice, cells were made permeable for the DNA by heat shocking for 45 seconds at 42 °C and a further 3 minute incubation on ice. The samples were than rescued by adding 500µl preheated antibiotic free LB-medium and incubated for one hour at 37 °C while shaking for induction of the antibiotic resistance. The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates.
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| - | == Isolation of plasmid DNA by alkaline lysis (mini and maxiprep) ==
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| - | For analysis of ligations and transformations QIAprep Spin Kits (Qiagen, Hilden) were used following the manufacturer instructions. For miniprep a single colony was picked from a LB-agar plate or glycerol stock was used to inoculate 5 ml LB-medium with appropriate antibiotic for selection (100 µg/µl ampicillin, 50 µg/µl kanamycin, 35 µg/µl chloramphenicol). Bacteria were grown over night at 37 °C while shaking (200 rpm). By using 4 ml over night culture with this kit the yield was around 6-10 µg. For maxipreps the Qiagen CompactPrep Plasmid Maxi Kit was used following the instructions given by the instruction manual. In this case 250 ml LB-medium were inoculated and used for preparation of plasmid DNA. The routinely yield was 300-400 µg plasmid DNA. Purity and amount of DNA was analysed using a NanoDrop.
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| - | == Site-directed mutagenesis ==
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| - | For removal of unwanted Restriction Sites, a PCR-based site direct mutagenesis protocol was adapted from [http://www.stratagene.com/manuals/200518.pdf Stratgene]. Oligos were designed to have a high (>78°C) Tm ny applying the formula
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| - | <math>T_{m} = 81{,}5 + 0{,}41 * (%GC) - \frac{675}N - %mismatch</math>.
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| - | The following scheme was used for pipetting:
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| - | {| class="wikitable"
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| - | |- bgcolor=grey
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| - | ! height=20px, width=900px |
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| - | |-align="center"
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| - | | style="font-weight:bold;" | 1 µl template (10 ng)
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| - | |-align="center"
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| - | | style="font-weight:bold;" | 1 µl primer 1 (25µM)
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| - | |-align="center"
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| - | | style="font-weight:bold;" | 1 µl primer 2 (25µM)
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| - | |-align="center"
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| - | | style="font-weight:bold;" | 25 µl Mastermix 2x (Phusion)
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| - | |-align="center"
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| - | | style="font-weight:bold;" | 22 µl dH2O
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| - | |}
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| - | The PCR procedure was as follows:
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| - | {| class="wikitable"
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| - | |- bgcolor=grey
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| - | ! height=20px, width=300px | || width=300px| || width=300px|
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| - | |-align="center"
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| - | | style="font-weight:bold;" |Initiale denaturation || 95 °C, 30 sec || 1 cycle
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| - | |-style="height:20px"
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| - | |-align="center"
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| - | | style="font-weight:bold;" |denaturation || 95 °C, 30 sec
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| - | |-align="center"
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| - | | style="font-weight:bold;" |annealing || 58 °C, 30 sec
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| - | |-align="center"
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| - | | style="font-weight:bold;" |extension || 72 °C, 40sec/kb + 30sec || 16 cycles
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| - | |-align="center"
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| - | |-align="center"
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| - | | style="font-weight:bold;" |termination || 72 °C, 5 – 10 min || 1 cycle
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| - | |-align="center"
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| - | | style="font-weight:bold;" | || 4 °C || forever
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| - | |-style="height:20px"
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| - | |-
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| - | |}
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| - | After completion, 1µL of DpnI (NEB) was added and the mix was incubated for 1h at 37°C. Then, DH5alpha cells were transformed as described above.
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| - | == DNA synthesis ==
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| - | Oligos were designed using [http://baderlab.bme.jhu.edu/gd/ GeneDesign]. 15Bp were chosen as an overhang and 56 °C as an annealing temperature.
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