Editing SLIC Cloning (section)

== Competent cells ==

TOP10 (Invitrogen)&nbsp;&nbsp;<br/>&nbsp;Inoculate TOP10 in 20 mL LB-medium in a 100 mL sterile Erlenmeyer flask&nbsp;<br/>&nbsp;Incubate&nbsp; overnight at 37°C, 300 r.p.m.<br/>&nbsp;Use the pre-culture&nbsp; &nbsp;to inoculate four 500 mL sterile Erlenmeyer flasks, each containing 250 mL LB-medium<br/>&nbsp;Incubated at 37°C, 250 r.p.m, until OD600 = 0.5–0.6 is reached.&nbsp;<br/>&nbsp;Cool down cultures&nbsp; &nbsp;on ice for 5 min<br/>&nbsp;Spin down cells in autoclave tube at 4°C for 5 min at 4,000 g.&nbsp;<br/>&nbsp;Resuspend cell pellets&nbsp; carefully in a total volume of 80 mL of freshly prepared (from stock solutions) ice-cold TFB1 buffer<br/>&nbsp;34.4 mL ddH2O, 8 mL 300 mM potassium acetate pH 6.0 (adjusted with KOH), 1.6 mL 500 mM CaCl2, 8 mL 500 mM MnCl2, 16 mL 500 mM RbCl, 12 mL glycerol, sterile filtered&nbsp;

&nbsp;Spin cells were for&nbsp; 5 min at 4°C at 2,000 g and discard the supernatant.<br/>&nbsp;Resuspend cells&nbsp; in a total volume of 8 mL of freshly prepared (from stock solutions) ice-cold TFB2 buffer&nbsp;<br/>&nbsp;4.72 mL ddH2O, 0.8 mL 100 mM MOPS pH 7.0, 1.2 mL 500 mM CaCl2, 80 μL 1M NaCl, 1.2 mL glycerol, sterile filtered<br/>&nbsp;Incubate&nbsp; on ice for 15 min.&nbsp;<br/>&nbsp;Transfer aliquots of 25 μL to sterile 1.5 mL Eppendorf tubes aand store&nbsp; at -80°C for further use
[[Category:Protocols]]