Team:HKUST/Protocols/plasmid extraction

Plamid DNA extraction

Purpose: To extract plasmid DNA from E.coli.

Materials: MX1 Buffer, MX2 Buffer, MX3 Buffer, WN Buffer, WS Buffer, Elution Buffer (all provided in Mini Plus Plasmid DNA Extraction System)

Procedure:

a.Grow 1 to 5 ml plasmid-containing bacterial cells in LB medium with appropriate anlibiotic overnight (12-16 hours) with vigorous agitation.
b. Pellet the cells by centrifuging for 1-2 minutes. Decant the supernatant and remove all medium residues by pipette.
c.Add 200 μL of MX1 Buffer 10 the pellet, and resuspend the cells completely by vortexing or pipetting.
d.Add 250 μL of MX2 Buffer and gently mix well (invert the tube 6-10 times) to lyse the cells until the lysate becomes d ear. Incubate at room temperature for 2-5 minutes.
e.Add 350 μL of MX3 Buffer to neutralize the lysate, then immediately and gently mix the solution well. A white precipitate should be formed.
f.Centrifuge at 10,000 x g (13,000rpm) for 5-10 minutes, meanwhile place a Mini Plus™ Column onto a Collection Tube. Transfer the supernatant carefully into the column.
g.Centrifuge at 7,000x g (9,000rpm) for 30-60 seconds. Discard the flow-through.
h.Wash the column once with 0.5 ml of WN Buffer by centrifuging at 7,000 x g (9,000rpm) for 30-60 seconds. Discard the flow-through.
i.Wash the column once with 0.7 ml of WS Buffer by centrifuging at 7,000 x g (9,000rpm) for 30-60 seconds. Discard the flow-through.
j.Centrifuge the column at 10,000 x g (13,000rpm) for another 3 minutes to remove residual ethanol.
k.Place the column into a new 1.5-ml centrifuge tube. Add 50 μL of Elution Buffer onto the center of the membrane.
l.Stand the column for 2-3 minutes, and centrifuge at 10,000 x g (13,000rpm) for 2-3 minutes to elute DNA. Store plasmid DNA at 4 °C or -20 °C.

Tips:

A. In step c, no clump should be visible after resuspension. Clumped cells lead to bad plasmid yield and quality.
B. Do not vortex in step d, otherwise genomic DNA will be sheared and contaminate the sample, which can be observed as serious foaming.
C. In step d, the lysate should become clear and viscous. Insufficient cell-lysis leads to low plasmid yield and quality.
D. MX1 Buffer must be stored at 4 °C.

• Agarose gel preparation and gel electrophoresis
• PCR (Taq, Vent)
• PCR product clean-up
• Enzyme digestion (vector, insert)
• Plasmid DNA extraction
• Gel cutting
• Gel extraction
• Ethanol precipitation
• Ligation
• Transformation to E.coli
• Transformation to yeast
• Cell lysis
• Yeast genomic DNA extraction
• Western blotting
• GFP testing