IGEM:Yale/2010/Protocols/miniprep

Standard Miniprep Protocols

We are using QIAprep Spin Miniprep Kits according to manufacturer instructions. These protocol are very slightly modified versions of what appears in the QIAprep Miniprep Handbook, 2nd ed.

Vacuum Manifold Protocol

1. Centrifuge sample in Eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution.
2. Resuspend pelleted bacterial cells in 250 μL of Buffer P1.
3. Add 250 μL Buffer P2 and mix thoroughly by inverting the tube 4-6 times.
4. Add 350 μL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
5. Centrifuge for 10 min at 13,000 rpm (approximately 17,900 times g) in a table-top microcentrifuge.
6. Apply the supernatant (from step 5) to the QIAprep spin column by decanting or pipetting.
7. Switch on the vacuum source to draw the solution through the QIAprep spin columns, and then switch off vacuum source.
8. Wash the QIAprep spin column by adding 0.5 mL Buffer PB. Switch on the vacuum source. After the solution has moved through the columns, switch off vacuum source.
9. Wash the QIAprep spin column by adding 0.75 mL Buffer PE. Switch on the vacuum source to draw the wash solution through the column,and then switch off vacuum source.
10. Transfer the QIAprep spin columns to micro centrifuge tubes. Centrifuge for 1 minute to remove residual wash buffer.
11. To elute DNA, place the QIAprep column in a clean 1.5 mL microcentrifuge tube. Add 30 μL of water to the center of each QIAprep spin column, let stand for 1 minute, and centrifuge 1 minute.
12. Rinse column by adding 10 μL of water to the center, letting it stand for one minute, and centrifuging for another minute.

Microcentrifuge Protocol

1. Centrifuge sample in Eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution.
2. Resuspend pelleted bacterial cells in 250 μL of Buffer P1.
3. Add 250 μL Buffer P2 and mix thoroughly by inverting the tube 4-6 times.
4. Add 350 μL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
5. Centrifuge for 10 min at 13,000 rpm (approximately 17,900 times g) in a table-top microcentrifuge.
6. Apply the supernatant (from step 5) to the QIAprep spin column by decanting or pipetting.
7. Centrifuge for 30-60 seconds. Discard flow-through.
8. Wash the QIAprep spin column by adding 0.5 mL Buffer PB and centrifuging for 30-60 seconds. Discard the flow-through.
9. Wash QIAprep spin column by adding 0.75 mL Buffer PE and centrifuging for 30-60 seconds.
10. Discard the flow-through, and centrifuge for an additional 1 minute to remove residual wash buffer.
11. To elute DNA, place the QIAprep column in a clean 1.5 mL microcentrifuge tube. Add 30 μL of water to the center of each QIAprep spin column, let stand for 1 minute, and centrifuge 1 minute.
12. Rinse column by adding 10 μL of water to the center, letting it stand for one minute, and centrifuging for another minute.

DNA not being put to use soon (in the next couple of days) should be stored at -20°C in our designated drawer.