Team:USTC/Experiments
Protocols
Plasmid Extraction
Materials :
Buffer P1 (stored in 4 degree)
Buffer P2
Buffer p3
VisualLyse
Buffer DW1
Wash Solution dd
ddH2O
Prepation
1.Check out whether RNaseA has been added in Buffer P1.
2.Check out whether ethyl alcohol has been added in Wash Solution
3.Check out whether sediment exist in Buffer P2 and P2. Procedure
4.Absorb 1.5 to 5 mL bacteria solution in to EP tubes, centrifuge them at 8,000xg 2 mins and then discard the culture medium.
5.Add 250 ul Buffer P1 into sediment, and use spearhead to make bacteria suspended.
6.Add 250 ul Buffer P2, and overturn the EP tubes 5 to 10 times immediately and tenderly. Stand tubes about 2 to 4 mins.
7.Add 350 ul Buffer P3 and overturn the EP tubes 5 to 10 times again immediately and tenderly.
8.Centrifuge tubes at 12,000xg about 5 to 10 mins.
9.Pour the supernatant liquid into absorption columns ,centrifuge them at 8,000xg about 30 sec and then discard the liquid in the collecting pipe.
10.optional:Add 500 ul Buffer DW1 and centrifuge them at 9,000xg about 30 sec. Then discard the liquid in the collecting pipe.
11.Add 500 ul Wash Solution, centrifuge them at 9,000xg about 30 sec and discard the liquid in the collecting pipe. 12.Do the step 11 again. 13.Centrifuge the empty columns at 9,000xg about 1 min. 14.Put the columns in clean 1.5mL EP tubes, add 50 to 100 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 9,000xg. 15.Keep the DNA solution for further work. The Protocol is based on SanPrep Column Type DNA Plasmid Extraction Kit.
Materials
