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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 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) |
| 25 µl Mastermix 2x (Phusion) |
| 22 µl dH2O |
The PCR procedure was as follows:
| Initiale denaturation | 95 °C, 30 sec | 1 cycle |
| denaturation | 95 °C, 30 sec | |
| annealing | 58 °C, 30 sec | |
| extension | 72 °C, 40sec/kb + 30sec | 16 cycles |
| termination | 72 °C, 5 – 10 min | 1 cycle |
| 4 °C | forever | |
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.
DNA synthesis
Oligos were designed using GeneDesign. 15Bp were chosen as an overhang and 56 °C as an annealing temperature. Oligos were ordered at a concentration of 100µM. 1:10 dilutions of the first and last oligo were prepared. Then, all oligos (including first and last) were pooled at 1µL each. Water was added to 10x the original volume. (So if a gene is synthesized from x = 14 oligos, water was added to 10*x = 140µL). This pool was then diluted 1:10. x µL of the dilution were put into a PCR reaction with 25µL Phusion master mix and 25-x µL water. PCR was conducted as following:
| Initiale denaturation | 95 °C, 5 min | 1 cycle |
| denaturation | 95 °C, 30 sec | |
| annealing | 58 °C, 30 sec | |
| extension | 72 °C, 1 minute | 7 cycles |
| 4 °C | forever | |
Afterwards, 1µL of the 1:10 dilutions of the innermost and outermost primer were added. The same PCR protocol was then repeated, but with 25 instead of 7 cycles. PCR products were run on a 2% agarose gel and gel purified.
Cell Culture
Every 3-4 days HeLa cells, MCS7 and U20S were splitted 1:3. Therefore DEMED medium (containing additionally 10% FCS, Pen-Strep, glutamate and non-essential aminoacids) was removed. Cells were washed afterwards with HBSS and trypsinised with 1 ml of trypsin and incubated for 5 min. at 37°C 5% CO2. The used flasks were filled up to a final volume of 7 ml with DMEM. 2 ml of the suspension was transfered to a new flask, 5 ml DMEM was added and incubated for another 3-4 days at 37°C and 5% CO2
