High School

**A. Preparation of Bacterial Culture**

1. Streak glycerol stock of *Agrobacterium tumefaciens* GV 2260 (RCAMBIA 2301) onto LB plates containing kanamycin (50 μg/ml).
2. Incubate at 28°C for two days.
3. Isolate a single colony and culture in 5 ml LB liquid medium containing kanamycin (50 μg/ml).
4. Incubate at 28°C overnight.

**B. Harvesting Bacterial Cells for Isolation of Plasmid DNA (RCAMBIA 2301)**

1. Centrifuge bacterial suspension (5 ml) at 4,000 g using a centrifuge for 15 minutes at room temperature.
2. Dispose of the supernatant into a waste beaker.

**C. Plasmid Isolation Using Qiagen Spin Miniprep Kit**

1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 (containing RNase A 25 μg) and transfer to a microcentrifuge tube. [Concentration of RNase A stock = 100 mg/ml]
2. Add 250 μl Buffer P2 and gently invert the tube 4-6 times to mix. **DO NOT VORTEX and DO NOT ALLOW THE LYSIS REACTION TO EXCEED MORE THAN 5 MINUTES.** [Time of lysis reaction should be 4 minutes]
3. Add 350 μl Buffer N3 and invert the tube immediately but gently 4-6 times to mix. The solution should become cloudy.
4. Centrifuge for 10 minutes at 13,000 rpm (17,900 g) in a tabletop microcentrifuge. A compact white pellet will form.
5. Apply the supernatant from step 4 to a QIAGEN spin column by decanting or pipetting.
6. Centrifuge for 30-60 seconds. Discard the flow-through.
7. Wash the QIAGEN spin column by adding 0.75 ml Buffer PE and centrifuge for 30-60 seconds.
8. Discard the flow-through and centrifuge for an additional 1 minute to remove residual wash buffer. **Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before the additional centrifugation. Residual ethanol from buffer PE may inhibit subsequent enzymatic reactions.**
9. Place the QIAGEN column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 minute and centrifuge for 1 minute.
10. Store plasmid DNA at -20°C. **(Please label isolated DNA with name and date.)**

Answer :

Final answer:

The preparation of bacterial culture and isolation of plasmid DNA involves streaking a glycerol stock of Agrobacterium tumefaciens GV 2260 (RCAMBIA 2301) onto LB plates, culturing a single colony in LB liquid medium, harvesting the bacterial cells, and performing plasmid isolation using the Qiarrer Spin Miniprep Kit. The Qiarrer Spin Miniprep Kit provides a quick and efficient method to obtain high-quality plasmid DNA from bacterial cultures.

Explanation:

To prepare a bacterial culture and isolate plasmid DNA, follow the steps below:

  1. Streak a glycerol stock of Agrobacterium tumefaciens GV 2260 (RCAMBIA 2301) onto LB plates containing kanamycin (50 ug/ml) and incubate at 28°C for two days.
  2. Isolate a single colony from the streaked plate and culture it in 5 ml LB liquid medium containing kanamycin (50 ug/ml) at 28°C overnight.
  3. Harvest the bacterial cells by centrifuging the 5 ml bacterial suspension at 4,000 g for 15 minutes at room temperature. Dispose of the supernatant.
  4. Perform plasmid isolation using the Qiarrer Spin Miniprep Kit:
  5. Resuspend the pelleted bacterial cells in 250 ul Buffer P1 (containing RNase A 25 g) and transfer to a microcentrifuge tube.
  6. Add 250 ul Buffer P2 and gently invert the tube 4-6 times to mix. Do not vortex and do not allow the lysis reaction to exceed more than 5 minutes.
  7. Add 350 ul Buffer N3 and invert the tube immediately but gently 4-6 times to mix. The solution should become cloudy.
  8. Centrifuge for 10 minutes at 13,000 rpm (17,900 g) in a table-top microcentrifuge. A compact white pellet will form.
  9. Apply the supernatant from step 4 to a QLARIER spin column by decanting or pipetting.
  10. Centrifuge the column for 30-60 seconds and discard the flow-through.
  11. Wash the QIARIER spin column by adding 0.75 ml Buffer PE and centrifuge for 30-60 seconds. Discard the flow-through.
  12. Centrifuge the column for an additional 1 minute to remove residual wash buffer. Residual ethanol from buffer PE may inhibit subsequent enzymatic reactions.
  13. Place the QLARIER column in a clean 1.5 ml microcentrifuge tube.
  14. To elute DNA, add 50 ul Buffer EB (10 mm Tris.CI. pH 8.5) or water to the center of each QIA prep spin column, let stand for 1 minute, and centrifuge for 1 minute.
  15. Store the isolated plasmid DNA at -20°C, labeled with the name and date.

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