SPEEDTOOLS TISSUE DNA EXTRACTION KIT - Biotools

2 PRE-LYSISThere are two possible working procedures:A) Resuspend the first pellet in 180 µl Buffer BT1 and 25 µlProteinase K. Transfer the resuspended solution of the firsttube to the second tube and the resuspended solution ofthe second tube to the third tube and so on. Finallycontinue with Step 3.B) Resuspend each pellet as mentioned above and proceedwith Step 3. In this case the solutions are pooled and thespin column has to be loaded successively.3 SAMPLE LYSISAdd 200 µl of lysis Buffer BB3 and incubate at least 20 min at70°C at least for 20 min. Vortex briefly.4 ADJUST DNA BINDING CONDITIONSAdd 210 µl of ethanol (96-100%) to the sample and vortexvigorously.+180 µlBUFFER BT1+25 µlPROTEINASE K200 µlBUFFER BB3Vortex and Incubate70°C, 20 min+210 µlETHANOLVortex5 BIND DNAFor each sample, place one column into a 2 ml collecting tubeand apply the sample to the column. Centrifuge at 4,500 × gduring 1 min. Discard the flow-through and place the columnback into the collecting tube.Load lysateinto a column1 min, 4,500 × g6 WASH SILICA MEMBRANE1 st WashAdd 500 µl Buffer BBW. Centrifuge 1 min at 4,500 x g. Discardflow-through and place the column back into the collecting tube.2 nd WashAdd 600 µl Buffer BB5. Centrifuge 2 min at 11,000 x g. Discardthe flow-through and place the column back into the collectingtube7 DRY SILICA MEMBRANEIncubate with open lid for 1-2 min at 70ºC. Residual ethanol isremoved during this step.8 ELUTE HIGHLY PURE DNAAdd 70 µl of pre-warmed elution Buffer BBE (70°C) directly ontothe silica membrane close the lid and incubate for further 3-5min at 70ºC. Centrifuge 1 min at 4,500 x g.+500 µlBUFFER BBW1 min, 4,500 × g+600 µlBUFFER BBW2 min, 11,000 ×gIncubate Tubeswith open lid1 min, 11,000 × g+70 µlBUFFER BBE(70ºC)Incubate 70ºC3-5 min1 min, 4,500 × gEd 05 – March 12Page 13 of 19SPEEDTOOLS TISSUE DNA EXTRACTION KIT