Team:University of Chicago/Notebook/DNA mini-prep

From 2008.igem.org

Latest revision as of 18:56, 29 July 2008

Zyppy mini-prep

The following procedure is performed at room temperature. Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion).

1. Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube
   • The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: 1A) Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. 1B) Discard the supernatant. 1C) Repeat steps 1A and 1B as needed. 1D) Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely.
2. Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.
3. Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.
4. Centrifuge at 11,000 – 16,000 x g for 2-4 minutes.
5. Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.Avoid disturbing the cell debris pellet.
6. Place the column into a Collection Tube and centrifuge for 15 seconds.
7. Discard the flow-through and place the column back into the same Collection Tube.
8. Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube.
9. Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds.
10. Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature.
11. Centrifuge for 15 seconds to elute the plasmid DNA.

Notes

1. Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been

neutralized and mixed thoroughly.

1. Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash

1. The Zyppy Elution Buffer cProxy-Connection: keep-alive

Cache-Control: max-age=0

tains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral pH) can also be used to elute the DNA.

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