TITLE: Insert + Vector DNA Ligation
AUTHORS: iGEM Dusseldorf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Pipette mix the following components:</div><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><td>H</td></tr><tr><td style = "text-align:center;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">Double Digested DNA</td><td rowspan = "" colspan = "" style ="display : table-cell;">ng/uL</td><td rowspan = "" colspan = "" style ="display : table-cell;">size (kb)</td><td rowspan = "" colspan = "" style ="display : table-cell;">bp ratio</td><td rowspan = "" colspan = "" style ="display : table-cell;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">3</td><td rowspan = "" colspan = "" style ="display : table-cell;">5</td><td rowspan = "" colspan = "" style ="display : table-cell;">I:V Ratio</td></tr><tr><td style = "text-align:center;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;">AXXX Vector, linearised + dephos</td><td rowspan = "" colspan = "" style ="display : table-cell;"> </td><td rowspan = "" colspan = "" style ="display : table-cell;"> </td><td rowspan = "" colspan = "" style ="display : table-cell;">#VALUE!</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">#VALUE!</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">ng insert</td></tr><tr><td style = "text-align:center;">3</td><td rowspan = "" colspan = "" style ="display : table-cell;">AXXX Gen, digested</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;">#VALUE!</td><td rowspan = "" colspan = "" style ="display : table-cell;"></td><td rowspan = "" colspan = "" style ="display : table-cell;">uL insert</td></tr><tr><td style = "text-align:center;">4</td><td rowspan = "" colspan = "" style ="display : table-cell;">Volume (uL)</td><td rowspan = "" colspan = "" style ="display : table-cell;">Ratio</td><td rowspan = "" colspan = "" style ="display : table-cell;">Insert</td><td rowspan = "" colspan = "" style ="display : table-cell;">Vector</td><td rowspan = "" colspan = "" style ="display : table-cell;">T4 Ligase</td><td rowspan = "" colspan = "" style ="display : table-cell;">Water</td><td rowspan = "" colspan = "" style ="display : table-cell;">T4 Buffer</td><td rowspan = "" colspan = "" style ="display : table-cell;">Total</td></tr><tr><td style = "text-align:center;">5</td><td rowspan = "" colspan = "" style ="display : table-cell;">Ligation 1 Vector only</td><td rowspan = "" colspan = "" style ="display : table-cell;">vector</td><td rowspan = "" colspan = "" style ="display : table-cell;">0</td><td rowspan = "" colspan = "" style ="display : table-cell;"> </td><td rowspan = "" colspan = "" style ="display : table-cell;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">17</td><td rowspan = "" colspan = "" style ="display : table-cell;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;">20</td></tr><tr><td style = "text-align:center;">6</td><td rowspan = "" colspan = "" style ="display : table-cell;">Ligation 2 Vector + Gene ligation</td><td rowspan = "" colspan = "" style ="display : table-cell;">3 to 1</td><td rowspan = "" colspan = "" style ="display : table-cell;">#VALUE!</td><td rowspan = "" colspan = "" style ="display : table-cell;"> </td><td rowspan = "" colspan = "" style ="display : table-cell;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">#VALUE!</td><td rowspan = "" colspan = "" style ="display : table-cell;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;">20</td></tr></table><div class = "text-block">Nur die grün markierten felder müssen ausgefüllt werden. Die Formeln berechnen das optimale 3:1 verhältnis von Vektor zu gen. Check wie viel DNA insgesammt eingesetzt wird. Vektor ist bei mir immer so um 25-30 ng und totale DNA menge überschreitet nicht 100 ng dann sollten gute ergebnisse erziehlt werden. </div><div class = "text-block"><span>Incubate 60 min at room temperature, and/or overnight at 16°C (</span><span style = "font-style:italic;">higher efficiency).</span><span style = "font-style:italic;"></span></div><div class = "text-block"><span style = "font-style:italic;">Optional</span><span>: inactivate ligase by incubating for 5 min in 70°C heat block.</span></div><div class = "text-block"><span style = "font-style:italic;">Transform into E</span><span>. coli</span></div></div>

[STEPS]