DNA试剂盒提取方法汉语版.docxVIP

This method allows genomic bacterial isolation from up to 3ml LB cultureMaterials and equipment to be supplied by user:Tabletop microcentrifugeNuclease-free 1.5ml microcentrifuge tubesWater bath capable of 37℃Shaking water bath capable of 55℃Incubator or water bath capable of 65℃100% ethanolIsopropanolTE bufferVortexerBefore startingPrepare DNA wash buffer.HBC buffer and lysozyme as instructed in the “preparing reagents” section on page 4Set an incubaor or water bath to 65℃Set a water bath to 37℃Set a shaking water bath to 55℃Heat elution buffer to 65℃culture bacteria in LB media to log-phase.(overnight culture can be used in many cases)Centrifuge no more than 3ml culture or 1×109 cells at 4000×g for 10 minutes at room temperatureAspiate and discard the mediaAdd 100μL TE buffer . vortex to complete resuspend the pelletAdd 10 μL lysozymeIncubate at 37℃ for 10 minutesNote: the amount of enzyme required and/or the length of incubation may need to be modified depending on the bacterial strain plete digestion of the cell wall is essential for efficient lysis.longer incubation time may yield better result.Optional:follow the short protocol belowfor difficult to lyse bacteria.add 25mg glass beads to 1.5ml microcentrifuge tube.Add sample to the glass beadsVortex at maximum speed for 5 minutesLet sample stang to allow the beads to settleTransfer supernatant to a new 1.5ml microcentrifuge tube.7.Add 100μL BTL buffer and 20μLProteinase K Solution.vortex to mix thoroughly.8.incubate at 55℃ in a shaking water bath.Note：usually no more than 1 hour is required for bacterial lysis.if a shaking water bath is not available,incubate the samples and shake or briefly vortex every20-30minutes.Add 5μL RNase A.INVERT TUBE SEVERAL TIMES TO MIXIncubate at room temperature for 5 minutesCentrifuge at 10000×g for 2 minutes to pellet any undigested materialTransfer the supernatan to a new 1.5ml microcentrifuge tube.do not disturb the pelletAdd 220μL BDL buffer . votex to mixIncubate at 65℃ for