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Minipreparation of plasmids

Purpose: Minipreparation (miniprep) is done in a laboratory setting in order to extract a plasmid from bacteria. There are several different scales of executing this task, but miniprep is the smallest. The steps of the miniprep are to weaken the cell wall, lyse the cell, precipitate out the lipids and proteins, remove of any chromosomal DNA or RNA that is still present, and then wash the plasmid to make sure that all salts are removed so that only the plasmid remains. This process uses a binding column that is comprised of a silica matrix in order to bind the plasmid DNA so that any other remaining cell fragments can be washed away. This procedure follows the E.Z.N.A. plasmid DNA Mini Kit 1 from the Omega Bio-Tek company.

Vacuum Procedure:

1. A culture was inoculated and grown overnight in a shaker at 37C at 300 rpm.

2. The culture was centrifuged at 10,000 rpm for 1 minute at room temperature

3. The culture median was decanted being careful not to discard any of the cell pellet.

4. 250 µL of Solution I was added and mixed thoroughly and resuspend the pellet. *Note: Solution I is used in order to weaken the cell wall.

5. After the pellet has been resuspended, the contents were added to a microcentrifuge tube.

6. 250 µL of Solution II was added and the microcentrifuge was gently inverted until a clear lysate formed. *Note: Solution II is used in order to lyse the cell wall. Vigorous mixing was avoided to prevent DNA shearing.

7. 350 µL of Solution III was added to the microcentrifuge tube and then it was inverted several times until a white precipitate forms. *Note: Solution III is used to bind all of the proteins and lipids from the cell so that they can be removed.

8. The solution was centrifuged at maximum speed for 10 minutes.

9. The cleared supernatant was transferred to a HiBind DNA Mini Column that was attached to the vacuum.

10. The vacuum was turned on and the liquid was drawn through the mini column.

11. 500 µL of HBC Buffer was added to the column and vacuumed through.

12. 700 µL of DNA wash buffer was added to the column and vacuumed through

13. Step 12 was repeated.

14. The column was added to a collection tube and then centrifuged for 2 minutes on maximum speed to dry the column.

15. The column was transferred to a clean 1.5 mL microcentrifuge tube.

16. 30 µL of sterile deionized water was added to the column and allowed to sit for 1 minute.

17. The microcentrifuge tube with the column inside of it was centrifuged for 1 minute at maximum speed.

18. The column was discarded and the microcentrifuge tube was stored at -20C until the plasmid was needed.