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=== 2. NucleoSpin® Plasma XS kit === : - Follow protocol described in [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-2-28#2._NucleoSpin.C2.AE_Plasma_XS_kit]] : - Add 1.35mL of Proteinase Buffer (PB) to 30mg of lyophilized Proteinase K for 50 prep kit. ==== Procedures ==== : # To remove residual cells, centrifuge plasma/serum at >11,000xg for 3 min<br> : <u>Preparing lysate</u> :: - Add '''200ul''' of '''plasma/serum/buffy coat''' to 1.5mL tube. :::{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''1X PBS (ul)''' |- | #1-P-Nu||100.00||100.00 |- | #1-S-Nu||200.00||0.00 |- | #1-BC-Nu||100.00||100.00 |- | #2-P-Nu||200.00||0.00 |- | #2-S-Nu||150.00||50.00 |- | #2-BC-Nu||100.00||100.00 |} :: - Add '''20ul''' of '''proteinase K''' to plasma/serum sample :: - Mix and incubate at 37C for 10min : <u>Binding DNA</u> :: - Add '''1.5 volume''' (300ul) of '''Buffer BB''' (keep sample:binding buffer ratio 1:1.5 v/v) :: - 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 '''26ul''' of '''Elution buffer''' to the column (range 5-30ul) :: - Centrifuge at 11,000xg for 30s ::: - Eluted DNA from buffy coat samples was red because erythrocytes were carried over with buffy coat layer either in sample #1 or #2. :: - Incubate eluted DNA with open lid at 90C for 8 min to remove residual ethanol --> ''I skipped this step by drying the column for 3 min before eluting DNA.'' ---- : - For bead-based method, I will split samples for two processes. One for regular DNA extraction and one will go to bisulfite treatment after DNA binding to beads without washing and elution. : - The volume of sample was not exactly 200ul (plasma and buffy coat ~100ul for each, serum ~200ul), so I used different volume for DNA extraction (50, 100 or 200ul)
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