Transformation of Bacteria by Plasmid DNA

[domba]

Supplies:

1. Microcentrifuge and adaptors for 600 ul tubes
2. Micropipetter and tips
3. Parafilm(R)
4. ice/ice water bath
5. Shaker at 37 degrees C
6. PCR machine and 600 ul tubes for use in the machine

Procedures:

1. Dilute the 2 ligation reactions 1:100 (to a concentration of 3-7 ng/ul of DNA) and place on ice.
2. Dilute pBC to a concentration of 1 ng/ul.
3. Thaw competent bacteria on ice.
4. Trasfer bacteria to 3 ice-cold 600 ul tubes.
5. Add 1 ul from each of the diluted ligation reactions and the diluted pBC.
6. Mix gently and incubate on ice for 30 minutes.
7. Heat the tubes at 42 degrees C for 20-30 seconds in the PCR machine.
8. Place on ice for 2 minutes.
9. Transfer bacteria to 3 test tubes.
10. Add 3 ml of LB medium.
11. Shake at about 150 rpm for 45 minutes at 37 degrees C.
12. Plate 100 ul of the bacteria onto one agar plate without antibiotic.
13. Plate 100 ul of the bacteria onto one agar plate with chloramphenicol.
14. Plate 200 ul of the bacteria onto another agar plate with chloramphenicol.
15. Invert the plates and place them at 37 degrees C overnight.
16. Seal the plates with Parafilm and store at 4 degrees C.

Results:

1. The agar plates without antibiotic are overgrown with bacteria the next day due to the lack of selection (such as the antibiotic chloramphenicol).
2. The agar plates with chloramphenicol are expected to have some white bacterial colonies and some blue one. The blue colonies are bacterial containing the vector DNA (without the insert). The white ones are bacteria containing ligated plasmid DNA with both vector and insert.
3. These plates can be stored at 4 degrees C for initiating overnight cultures later.