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==== Procedures ==== : # To remove residual cells, centrifuge plasma/serum at >11,000xg for 3 min<br> : <u>Preparing lysate</u> :: - Add '''240ul''' of '''plasma/serum''' to 1.5mL tube. --> ''' I will add 200ul''' :: - Add '''20ul''' of '''proteinase K''' to plasma/serum sample :: - Mix and incubate at 37C for 10min : <u>Binding DNA</u> :: - Add '''1.5 volume''' (360ul) of '''Buffer BB''' (keep sample:binding buffer ration 1:1.5 v/v) --> '''should be 300ul of Buffer BB''' :: - Invert the tube 3x and vortex for 3s. Spin down the tube briefly. :: - Load 600ul of the mixture to a Nucleospin Plasma XS column :: - Centrifuge at 2,000xg for 30s. Centrifuge at higher speed at 11,000xg for further 5s. : <u>Washing DNA</u> :: - Transfer Nucleospin column to a new collecting tube :: - Add '''500ul''' of '''buffer WB''' onto the column. Centrifuge at 11,000xg for 30s. :: - Place column into new collecting tube :: - Add '''250ul''' of '''buffer WB''' to the column. Centrifuge at 11,000xg for 3min : <u>Eluting DNA</u> :: - Place column to 1.5mL tube :: - Add '''20ul''' of '''Elution buffer''' to the column (range 5-30ul) :: - Centrifuge at 11,000xg for 30s :: - Incubate eluted DNA with open lid at 90C for 8 min to remove residual ethanol
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