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Team:Heidelberg/n4tr1um
From 2009.igem.org
Contents |
Transformation of Bacteria
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.
Isolation of plasmid DNA by alkaline lysis (mini and maxiprep)
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.
Site-directed mutagenesis
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 <math>T_{m} = 81{,}5 + 0{,}41 * (%GC) - \frac{675}N - %mismatch</math>. The following scheme was used for pipetting:
| 1 µl template (10 ng) |
| 1 µl primer 1 (25µM) |
| 1 µl primer 2 (25µM) |
| 0,5 µl polymerase (Phusion) |
| 1 µl dNTP mix (10 mM) |
| 10 µl 5x Phusion HF buffer |
| 38 µl dH2O |
The PCR procedure was as follows:
| Initiale denaturation | 95- 98 °C, 30 sec | 1 cycle |
| denaturation | 95-98 °C, 10 sec | |
| annealing/elongation | 72 °C, 30sec/1kb + 30sec | 25-28 cycles |
| termination | 72 °C, 5 – 10 min | 1 cycle |
| 4 °C | forever | |
DNA synthesis
Oligos were designed using [http://baderlab.bme.jhu.edu/gd/ GeneDesign]. 15Bp were chosen as an overhang and 56 °C as an annealing temperature.
