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== Column-based methods == === 1. QIAamp DNA Blood Mini Kit === : - Resuspend lyophilized QIAGEN Protease with 1.2 ml protease solvent. : - Use carrier DNA (e.g., poly dA, poly dT, poly dA:dT) when the sample is low-copy (i.e., when <10,000 copies are present) Starting material: 200ul for blood, plasma, serum or buffy coat ==== Procedures ==== : <u>Preparing lysate</u> :: - Pipet '''20 µl''' of '''QIAGEN Protease''' (or proteinase K) into the bottom of a 1.5 ml tube :: - Add '''200 µl sample''' to the tube. If the volume of sample less than 200ul, adjust volume with PBS :: - Add '''200 µl''' of '''Buffer AL''' to the sample. Mix by pulse-vortexing for 15 s :: - Incubate at 56°C for 10 min : <u>Binding DNA</u> :: - Briefly spin down the tube before adding 200ul '''100% EtOH'''. Mix again by pulse-vortexing for 15 s and briefly spin down :: - Transfer spnt to QIAamp Mini spin column & centrifuge at 6,000xg for 1 min : <u>Washing DNA</u> :: - Transfer QIAamp Mini spin column in a clean 2 ml collection tube :: - Add '''500 µl''' of '''Buffer AW1''' & centrifuge at 6,000xg for 1 min :: - Transfer QIAamp Mini spin column in a clean 2 ml collection tube :: - Add '''500 µl''' of '''Buffer AW2''' & centrifuge at 20,000xg for 3min : <u>Eluting DNA</u> :: - Transfer the QIAamp Mini spin column in a new 2 ml collection tube and centrifuge at 20,000xg for 1 min (full speed) :: - Place the QIAamp Mini spin column in a clean 1.5 ml tube & add appropriate volume of '''Buffer AE''' or distilled water (manual recommends 200ul). :: - Incubate at RT for 5 min & centrifuge at 6,000xg for 1 min <br> === 2. NucleoSpin® Plasma XS kit === '''Product specifications''' Starting material: Up to 240 μL EDTA plasma Elution volume: 5–30 μL Average yield: 0.1–100 ng per mL plasma DNA size range: 50-1000bp DNA yield from human plasma: DNA amounts from less than 0.1 ng DNA per mL of plasma up to several 100 ng DNA per mL of plasma have been reported : - 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 '''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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