Here is a protocol that we use. It is a modified alkaline lysis method. I cannot remember the original source at this moment.

1. Centrifuge 1.5 mL of Agrobacterium culture in a 1.7 mL microfuge tube.

2. Resuspend pellet in 350 µL 1X TE. (note: use Qiagen P1 and if you are doing PCR or electroporation you can skip the RNASe A step)

3. Add 50 µL of 10% SDS.

4. Add 2 µL of 10 mg/mL Proteinase K.

5. Incubate for 1 hr at 37°C.

6. Add 50 µL of 5M NaCl. Mix well.

7. Incubate for 30 minutes at 68°C in a water bath.

8. Phenol/CHCl3 extract.

9. Phenol/CHCl3 extract again.

10. CHCl3 extract.

11. Add 903 µl of 95% (or 100%) ethanol (EtOH) and mix well.

12. Incubate at room temperature for 15 minutes.

If precipitated DNA mass is large, then picofuge to pellet. Otherwise, spin 10 min. at 10 Krpm.

13. Dissolve pellet in 200 µL of 1X TE.

14. Using a filter tip, add 10 µl of 1 mg/mL RNAse A. Mix well.

15. Incubate for 30 minutes at 37°C.

16. Transfer 100% of sample to a new, 0.6 mL microfuge tube.

17. Phenol/CHCl3 extract.

18. Phenol/CHCl3 extract again.

19. CHCl3 extract.

20. Add 20 µl of 3M NaOAc pH 6- 7

21. Add 440 µl of 95- 100% etOH. Mix really, really well.

22. Incubate at room temperature for 15 minutes.

If precipitated DNA mass is large, then picofuge to pellet. Otherwise, spin 10 min. at 10 Krpm.

23. Dissolve pellet in 100 µL 1X TE.

24. Store DNA at 4 °C.

Use 1 µl of purified plasmid DNA to electroporate into appropriate E. coli competent cells, if DNA isolation was for the purpose of clone verification. Typically, plating 10 µl of transformed cells should be enough for many colonies, depending of course on how competent your E. coli cells are.

Good luck!

Hello Vinoth,

It is indeed difficult to get good quality plasmid from agrobacterium clones. So what is advisable would be to isolate your plasmid simply by the alkaline lysis method or any column based kit and then transform the plasmid back into any E. coli strain and then isolate it from there. This is what we practice in our lab. However, since you need it for PCR analysis i think a dilution of the agrobacterium isolated plasmid should work for a PCR reaction. but it does not give the restriction profile and for that you have to do the back transformation.

Thank you Ms.Harmeet Kaur! I have tried alkaline lysis method but i could not observe the band for plasmid in agarose gel

Here is a protocol that we use. It is a modified alkaline lysis method. I cannot remember the original source at this moment.

1. Centrifuge 1.5 mL of Agrobacterium culture in a 1.7 mL microfuge tube.

2. Resuspend pellet in 350 µL 1X TE. (note: use Qiagen P1 and if you are doing PCR or electroporation you can skip the RNASe A step)

3. Add 50 µL of 10% SDS.

4. Add 2 µL of 10 mg/mL Proteinase K.

5. Incubate for 1 hr at 37°C.

6. Add 50 µL of 5M NaCl. Mix well.

7. Incubate for 30 minutes at 68°C in a water bath.

8. Phenol/CHCl3 extract.

9. Phenol/CHCl3 extract again.

10. CHCl3 extract.

11. Add 903 µl of 95% (or 100%) ethanol (EtOH) and mix well.

12. Incubate at room temperature for 15 minutes.

If precipitated DNA mass is large, then picofuge to pellet. Otherwise, spin 10 min. at 10 Krpm.

13. Dissolve pellet in 200 µL of 1X TE.

14. Using a filter tip, add 10 µl of 1 mg/mL RNAse A. Mix well.

15. Incubate for 30 minutes at 37°C.

16. Transfer 100% of sample to a new, 0.6 mL microfuge tube.

17. Phenol/CHCl3 extract.

18. Phenol/CHCl3 extract again.

19. CHCl3 extract.

20. Add 20 µl of 3M NaOAc pH 6- 7

21. Add 440 µl of 95- 100% etOH. Mix really, really well.

22. Incubate at room temperature for 15 minutes.

If precipitated DNA mass is large, then picofuge to pellet. Otherwise, spin 10 min. at 10 Krpm.

23. Dissolve pellet in 100 µL 1X TE.

24. Store DNA at 4 °C.

Use 1 µl of purified plasmid DNA to electroporate into appropriate E. coli competent cells, if DNA isolation was for the purpose of clone verification. Typically, plating 10 µl of transformed cells should be enough for many colonies, depending of course on how competent your E. coli cells are.

Good luck!