Maheshri:MiniprepY: Difference between revisions
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[http:// | '''<font size = "+1">Yeast Plasmid Prep</font>''' | ||
The following protocol is based on the [http://openwetware.org/wiki/Miniprep/Qiagen_kit Qiagen miniprep kit]. | |||
#Inoculate a single yeast colony into 2–5 ml of the appropriate selective media and grow the culture for 16–24 h at 30°C. | |||
#Harvest the cells by centrifugation for 5 min at 5000 x g and resuspend cells in 250 μl Buffer P1 containing 0.1 mg/ml RNase A. Transfer the cell suspension to a 1.5 ml microcentrifuge tube. | |||
#Add 50–100 μl of acid-washed glass beads (Sigma G-8772) and vortex for 5 min. Let stand to allow the beads to settle. Transfer supernatant to a fresh 1.5 ml microcentrifuge tube. | |||
#Add 250 μl lysis buffer P2 to the tube and invert gently 4–6 times to mix. Incubate at room temperature for 5 min. | |||
#Add 350 μl neutralization buffer N3 to the tube and invert immediately but gently 4–6 times. | |||
#Centrifuge the lysate for 10 min at maximum speed in a tabletop microcentrifuge (13,000 rpm or ≥10,000 x g). Meanwhile, place a QIAprep Spin Column in a 2 ml collection tube. | |||
#Transfer the cleared lysate from step 6 to QIAprep Spin Column by decanting or pipetting. | |||
#Centrifuge for 30–60 s (13,000 rpm or ≥10,000 x g). Discard flow-through. | |||
#Wash QIAprep Spin Column by adding 0.75 ml of Buffer PE and centrifuging 30–60 s (13,000 rpm or ≥10,000 x g). | |||
#Discard flow-through and centrifuge for an additional 1 min to remove residual wash buffer (13,000 rpm or ≥10,000 x g). IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. | |||
#Place QIAprep Spin Column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 25 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep Spin Column, let stand for 1 min, and centrifuge for 1 min. For subsequent transformation into E. coli, 2–3 μl of eluate yields about 10 colonies (using chemically competent cells with an efficiency of 10<sup>7</sup>). | |||
Latest revision as of 18:29, 7 September 2009
Yeast Plasmid Prep
The following protocol is based on the Qiagen miniprep kit.
- Inoculate a single yeast colony into 2–5 ml of the appropriate selective media and grow the culture for 16–24 h at 30°C.
- Harvest the cells by centrifugation for 5 min at 5000 x g and resuspend cells in 250 μl Buffer P1 containing 0.1 mg/ml RNase A. Transfer the cell suspension to a 1.5 ml microcentrifuge tube.
- Add 50–100 μl of acid-washed glass beads (Sigma G-8772) and vortex for 5 min. Let stand to allow the beads to settle. Transfer supernatant to a fresh 1.5 ml microcentrifuge tube.
- Add 250 μl lysis buffer P2 to the tube and invert gently 4–6 times to mix. Incubate at room temperature for 5 min.
- Add 350 μl neutralization buffer N3 to the tube and invert immediately but gently 4–6 times.
- Centrifuge the lysate for 10 min at maximum speed in a tabletop microcentrifuge (13,000 rpm or ≥10,000 x g). Meanwhile, place a QIAprep Spin Column in a 2 ml collection tube.
- Transfer the cleared lysate from step 6 to QIAprep Spin Column by decanting or pipetting.
- Centrifuge for 30–60 s (13,000 rpm or ≥10,000 x g). Discard flow-through.
- Wash QIAprep Spin Column by adding 0.75 ml of Buffer PE and centrifuging 30–60 s (13,000 rpm or ≥10,000 x g).
- Discard flow-through and centrifuge for an additional 1 min to remove residual wash buffer (13,000 rpm or ≥10,000 x g). IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
- Place QIAprep Spin Column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 25 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep Spin Column, let stand for 1 min, and centrifuge for 1 min. For subsequent transformation into E. coli, 2–3 μl of eluate yields about 10 colonies (using chemically competent cells with an efficiency of 107).
