TITLE: Natural competence of Bacillus subtilis transformation
AUTHORS: iGEM Dusseldorf

[DESCRIPTION]
<div class = "text-blocks"><table border><tr style = "text-align:center;"><td>  </td><td>A</td><td>B</td><td>C</td><td>D</td><td>E</td><td>F</td><td>G</td></tr><tr><td style = "text-align:center;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">final concentration</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">Stock</td><td rowspan = "" colspan = "" style ="display : table-cell;">for 50 ml</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">for 20 ml</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td></tr><tr><td style = "text-align:center;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;">100 mM Kaliumphosphatpuffer pH 7 :</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;"></td></tr><tr><td style = "text-align:center;">3</td><td rowspan = "" colspan = "" style ="display : table-cell;">60 mM K2HPO4</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">1 M (3,48 g in 20 ml for Stocklösung)</td><td rowspan = "" colspan = "" style ="display : table-cell;">3 ml</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td><td rowspan = "" colspan = "" style ="display : table-cell;">1,2 ml</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td></tr><tr><td style = "text-align:center;">4</td><td rowspan = "" colspan = "" style ="display : table-cell;">40 mM KH2PO4</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">0,5 M (1,36 g in 20 ml for Stocklösung)</td><td rowspan = "" colspan = "" style ="display : table-cell;">4 ml</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td><td rowspan = "" colspan = "" style ="display : table-cell;">1,6 ml</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td></tr><tr><td style = "text-align:center;">5</td><td rowspan = "" colspan = "" style ="display : table-cell;">3 mM Trinatriumcitrat</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">0,5 M (0,735 g in 5 ml)</td><td rowspan = "" colspan = "" style ="display : table-cell;">300 µl</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td><td rowspan = "" colspan = "" style ="display : table-cell;">120 µl</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td></tr><tr><td style = "text-align:center;">6</td><td rowspan = "" colspan = "" style ="display : table-cell;">20 mM Kalium-L-Glutamat</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">1 M (4,06 g in 20 ml)</td><td rowspan = "" colspan = "" style ="display : table-cell;">1 ml</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td><td rowspan = "" colspan = "" style ="display : table-cell;">400 µl</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td></tr><tr><td style = "text-align:center;">7</td><td rowspan = "" colspan = "" style ="display : table-cell;">21 mM MgSO4</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">1 M (1,23 g in 5 ml)</td><td rowspan = "" colspan = "" style ="display : table-cell;">1050 µl (7x)</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td><td rowspan = "" colspan = "" style ="display : table-cell;">420 µl (7x)</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td></tr><tr><td style = "text-align:center;">8</td><td rowspan = "" colspan = "" style ="display : table-cell;">1 % Glukose</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">50 % (10g in 20 ml)</td><td rowspan = "" colspan = "" style ="display : table-cell;">1 ml</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td><td rowspan = "" colspan = "" style ="display : table-cell;">400 µl</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td></tr><tr><td style = "text-align:center;">9</td><td rowspan = "" colspan = "" style ="display : table-cell;">20 mg/ml L-Tryptophan</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">5 mg/ml (25 mg in 5 ml)</td><td rowspan = "" colspan = "" style ="display : table-cell;">200 µl</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td><td rowspan = "" colspan = "" style ="display : table-cell;">80</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td></tr><tr><td style = "text-align:center;">10</td><td rowspan = "" colspan = "" style ="display : table-cell;">0,1 % Caseinhydrolysat (DIFCO!)</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">10 % (1 g in 10 ml)</td><td rowspan = "" colspan = "" style ="display : table-cell;">500 µl</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td><td rowspan = "" colspan = "" style ="display : table-cell;">200 µl</td><td rowspan = "" colspan = "" style ="display : table-cell;">⃝</td></tr></table><div class = "text-block">Inoculation VK: Spread B. subtilis out of cryo on LB plate and apply üN 37°C or üW 30°C</div><div class = "text-block">Single clone in 5 ml Paris Medium (test tube) on incubator roller or plate washer with 1 ml Paris Medium (saves one day work)</div><div class = "text-block">Inoculation of 2,5 ml Paris Medium (test tube) from VK (dishwasher or liquid-ÜK) to OD580=0,2</div><div class = "text-block">4h incubation on incubator roller at 37°C</div><div class = "text-block">1 ml pellet cells in Eppi (1min, max, table centrifuge)</div><div class = "text-block">Resuspend the pellet in 1 ml Paris medium with 10 % (v/v) glycerol.</div><div class = "text-block">Store 100 µl aliquots at -80 °C</div><div class = "text-block">Thaw aliquots at 37 °C, add 900 µl Paris medium and 500-1000 ng plasmid DNA (test tube)</div><div class = "text-block">Incubate 6 h in the incubator roller at 37°C</div><div class = "text-block">Pellet cells, remove 800 µl, resuspend and plate the rest (for normal transformer on LB+antibiotic, for upp mutants FB medium with 5-fluorouracil)</div><div class = "text-block">concentrations of antibiotics: Kanamycin 50 yg/ml, Neomycin 5 yg/ml, Erytromycin 1 yg/ml, Tetracycline 20 yg/ml, Chloramphenicol 5 yg/ml, Linkomycin 25 yg/ml</div></div>

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