In the hood: 96 well dish with bacteria titertech microtubes glass pipette Remove colonies from each well using the titertech and place them into the cover Pipette up and down to thoroughly mix the colonies Aliquot 300 µl of the culture into a microtube; save about 4 or 5 tubes In the lab: Centrifuge the culture for about 2 minutes or until a pellet has formed Remove the supernatant Resuspend the pellet in 576 µl TE, 15 µl of 20% SDS and 3 µl of 20 mg/ml proteinase K Incubate for 1 hour at 37 ℃ Add 166 µl of 3M NaCl and mix thoroughly Add 80 µl of 10% CTAB in 0.7 M NaCl and thoroughly mix Incubate for 10 minutes at 65 ℃ In the hood in the back: Add an approximately equal volume of chloroform (700 µl) In the lab: Centrifuge for 5 minutes at room temperature In the hood in the back: Remove white interface (should be able to be done by using pipetter) Transfer supernatant to another microtube Discard remaining solution in chloroform waste receptacle Add an equal amount of phenol/chloroform In the lab: Centrifuge for 5 minutes In the hood in the back: Transfer supernatant to a new microtube Discard remaining solution in proper waste receptacle In the lab: Add 0.6 vol of isopropanol and gently rock back and forth until white precipitant forms Centrifuge for 30 minutes Remove supernatant and wash pellet with 70% ethanol Centrifuge the tube for 5 minutes Remove supernatant Redissolve the pellet in 100 µl TE/10 |
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