Team:SCU-China/Plasmid

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1.Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1-5 ml LB media containing the appropriate selective antibiotic. Incubate for 12-16 hr at 37℃ with vigorous shaking (about 300rpm). Use a 10-20 ml culture tube or a flask with a volume of at least 4 times the volume of the culture. It is strongly recommended that an endA negative strain of E.coli be used for routine plasmid isolation. Examples of such strains include DH5α and JM109.

2.Pellet 1.5-5.0 ml bacteria by centrifugation at 10000 x g for 1 min at room temperature. Decant or aspirate medium and discard.

3.Resuspend the bacterial pellet by adding 250 μl of Solution I/RNase A and vortexing (or pipetting up and down). Complete resuspension (no visible cell clumps) of cell pellet is vital for obtaining good yields.

4.Add 250 μl of Solution II and gently mix by inverting and rotating the tube several times to obtain a clear lysate. A 2 minutes incubation may be necessary. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 min. (Store Solution II tightly capped when not in use to avoid acidification from CO2 in the air.)

5.Add 350 μl of Solution III and mix immediately by inverting the tube several times until a flocculent white precipitate forms. It is vital that the solution is mixed thoroughly, and immediately after the addition of Solution III to avoid localized precipitation.

6.Centrifuge at ≥ 13000 x g for 10 minutes at room temperature. A compact white pellet will form. Promptly proceed to the next step.

7.Add the cleared supernatant by CAREFULLY aspirating it into a clean HiBind Miniprep Column I assembled in a provided 2 ml collection tube. Ensure that the pellet is not disturbed and that no cellular debris has been carried over into the column. Centrifuge for 1 min at 10000 x g at room temperature to completely pass lysate through the HiBind Miniprep Column I

8.Discard flow-through liquid and re-use the 2 ml collection tube. Add 500 μl of Buffer HB to wash the HiBind Mniprep Column I. Centrifuge for 1 min at 1000 x g at room temperature to completely pass solution through the HiBind Miniprep Column I. This step ensures that residual protein contaminations are remved, thus ensuring high quality DNA that will be suitable for downstream applications.

9.Discard flow-through liquid and re-use the 2 ml collection tube. Add 700 μl of DNA Wash Buffer diluted with absolute ethanol to wash the HIBind Miniprep Colun I. Centrifuge for 1 min at 10000 x g at room temperature to completely pass solution through the HiBind Miniprep Column I and discard flow-through liquid.

Note: DNA Wash Buffer concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated, DNA Wash Buffer must be brought to room temperature before use.

Optional Step: Repeat wash step with another 700 μl of DNA Wash Buffer diluted with absolute ethanol.

Centrifuge the empty column for 2 min at ≥ 13000 x g to dry the column matrix. Do not skip this step, it is critical for good yields.

Place the column into a clean 1.5 ml micro-centrifuge tube. Add 30 μl to 50 μl (Depending on desired concentration of final product) of Elution Buffer (10mM Tris-HCl, PH 8.5) or sterile deionized water directly onto the column matrix and let it sit at roon temperature for 1-2 minutes. Centrifuge for 1 min at ≥13000 x g to elute DNA. An optional second elution will yield any residual DNA, through at a lower concentration.