染色體DNA的萃取(傳統方法).docVIP

『分子基因選殖』實習手冊 （一）Mini-M plasmid DNA extraction system (VIOGENE) Grow 1 to 5ml plasmid-containing bacterial cells in LB medium with appropriate antibiotics overnight (12-16 hours) with vigorous agitation ↓ Pellet the cells by centrifuging for 1-2 minutes. Decant the supernatant and remove all medium residue by pipet. ↓ Add 250 ?l of MX1 Buffer to the pellet, and resuspend the cells completely by vortexing or pipetting. ↓ Add 250 ?l of MX2 Buffer and gently mix (Invert the tube 4 ~ 6 times) to lyse the cells until the lysate becomes Clear. Incubate at room temperature for 1-5 minutes. ↓ Add 350 ?l of MX3 Buffer to neutralize the lysate, then immediately and gently mix the solution. A White precipitate should be formed. ↓ Centrifuge for 5~10 minutes, meanwhile place a mini-M Column onto a Collection Tube. ↓ Transfer the supernatant carefully into the column ↓ Centrifuge for 30-60 seconds. Discard the flow-through. ↓ Wash the column once with 0.5 ml WF Buffer by centrifuging for 30-60 seconds. Discard the flow-through. ↓ Wash the column once with 0.7 ml WS Buffer by centrifuging for 30-60 seconds. Discard the flow-through. ↓ Centrifuge the column at full speed for another 3 minutes to remove residual ethanol. ↓ Place the column onto a new 1.5-ml centrifuge tube. Add 50μI of Elution Buffer (provided) onto the center of the membrane. ↓ Stand the column for 1-2 minutes, and centrifuge for 1-2 minutes to elute DNA. ↓ Store plasmid DNA at 4°C or -20°C. （二）Isolation of plasmid DNA (small-scale)(傳統方法) overnight culture 1.5 ml, 8000g, 5min, 4℃ ↓ leaving bacterial pellet as dry as possible ↓ resuspension pellet with 100 ?l ice-cold solution I (Gram positive adds lysozyme final 1 mg/ml, put 37℃ 1hr ) ↓ add 200 ?l solution II (fresh), iverting the tube rapid ↓ add 150 ?l solution III, mixing, light votex, on ice 3~5 min ↓ 12000g, 5 min 4℃, transfer suspension to fresh tube ↓ phenol (phenol-chloroforn) extraction unitl no white protein ↓ 12000g, 5 min 4℃, transfer upper layer to f