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==== Procedures ==== ''' Suggested reagent volumes for different sample volumes ''' :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Reagent\sample volume (S)''' | align="center" style="background:#f0f0f0;" width="60"|'''1mL''' | align="center" style="background:#f0f0f0;" width="60"|'''800ul''' | align="center" style="background:#f0f0f0;" width="60"|'''600ul''' | align="center" style="background:#f0f0f0;" width="60"|'''400ul''' | align="center" style="background:#f0f0f0;" width="60"|'''200ul''' |- | A volume: ChargeSwitch Lysis Buffer (L19)||700 ul||560 ul||420 ul||280 ul||140 ul |- | Proteinase K||30 ul||30 ul||30 ul||30 ul||30 ul |- | Rnase A (optional)||5 ul||5 ul||5 ul||5 ul||5 ul |- | B volume: ChargeSwitch Purification Buffer (N7)||250 ul||200 ul||150 ul||100 ul||50 ul |- | C volume: ChargeSwitch Magnetic beads||30 ul||24 ul||18 ul||12 ul||6 ul |- | D volume: ChargeSwitch Wash Buffer (W12)||1 mL ||800 ul||600 ul||400 ul||200 ul |- | ChargeSwitch Elution Buffer (E5)||50 ul||50 ul||50 ul||50 ul||50 ul |} : # Before starting, mix Lysis buffer with proteinase K according to table above & scale up volume for multiple samples : <u>Preparing lysate</u> :: - Add '''S volume''' of sample to 1.5mL tube :: - Add '''A volume''' + 30ul of '''Lysis mix''' (Lysis Buffer + proteinase K) :: - Add '''5ul''' of '''RNase A''' :: - Pipette up & down 5x to mix :: - Incubate at RT for 20min to lyse the sample. Alternatively incubate on a rotating mixer : <u>Binding DNA</u> : - Vortex the tube containing ChargeSwitch Magnetic beads :: - Add '''B volume''' of '''ChargeSwitch Purification Buffer''' to the digested sample :: - Add '''30 ul''' of '''ChargeSwitch Magnetic beads''' and pipette up & down 5x to mix (use p1000 pipette, avoid generating bubbles) :: - Incubate at RT for 2 min to allow DNA bind to magnetic beads :: - Place the sample tube in the MagnaRack for 3 min or until the bead forming a tight pellet. (I may use magnetic rack for 1.5mL tube available in the lab) :: - Carefully remove the spnt without disturbing the bead pellet : <u>Washing DNA</u> :: - Remove sample tube from magnetic rack :: - Add '''D volume''' of '''ChargeSwitch Wash Buffer''' to the tube and pipette up & down 5x gently & avoid bubbles :: - Place the sample tube in the MagnaRack for 2 min or until the bead forming a tight pellet :: - Carefully remove the spnt without disturbing the bead pellet :: - Repeat washing step : <u>Eluting DNA</u> :: - Remove sample tube from magnetic rack (there should be no spnt in the tube) :: - Add '''50ul''' of '''ChargeSwitch Elution Buffer''' to the tube and pipette up & down gently 10x to resuspend magnetic beads ::: # '''Do not use H2O for elution''' :: - Place the sample tube in the MagnaRack for 1 min or until the bead forming a tight pellet :: - Carefully transfer eluted DNA to 1.5mL tube
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