Transformation: competent DH5⍺ cells with ligation product BBA and BBB

Objectives

The objective is to transform competent DH5⍺ cells with the ligations products BBA and BBB.

Materials

• 2 aliquots of "old" DH5⍺ Competent cells (from the 23/07/16)
• 2 aliquots of "new" DH5⍺ Competent cells (from the 20/09/16)
• Plasmid DNA : Ligation product BBA and BBB (from the 19/09/16)
• Petri dish LB+Cm: Cm concentration = 25 µg/mL

Protocol

Experimental conditions realized :

Transformation protocol:

1. Thaw tubes of DH5⍺ competent cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into the 4 transformation tubes on ice.
2. Add the 10 µL plasmid DNA to the cell mixture.
3. Carefully flick the tubes 4-5 times to mix cells and DNA. Do not vortex.
4. Place on ice for 30 min. Do not mix.
5. Heat shock at exactly 42°C for 45 s. Do not mix.
6. Place on ice for 5 min. Do not mix.
7. Pipette 250 µL of room temperature SOC into the mixture.
8. Place at 37°C for 1h at 250 rpm.
9. Warm selection plates to 25°C.
10. Mix the cells thoroughly by flicking the tubes and inverting.
11. Spread the corresponding volume onto each plate.
12. Incubate all the plates O/N at 37°C.

Results (obtained on the 21/09)

Expected results:

Some colonies on the petri dishes LB+Cm plated with 50 µL of bacteria transformed with the different ligation products and more on the petri dishes LB+Cm plated with 200 µL of bacteria.
More colonies on the petri dishes plated with the « new » competent bacteria (from 20/09) transformed with the different ligation products.
A bacterial lawn on the LB petri dishes without antibiotic.
No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control).

Obtained results:

We obtained expected results.

Interpretation

The transformation worked. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. A PCR colonie is necessary to check the size of the plasmid present in colonies, and therefore in order to know if bacteria integrated the correct plasmid.

Competent cells: E.Coli DH5⍺

Objectives

The objective is to make a stock of E.Coli DH5⍺ competent cells for subsequent transformations.

Materials

• O/N DH5⍺ pre-culture (made the 22/07): O/N inoculation of 100 µL DH5⍺ into 100 mL LB.
• 0.1M CaCl2: prepared the 23/07
• 0.1M CaCl2/15% Glycerol: prepared on the 23/07

Protocol

Competence:

1. Inoculation of 3 mL O/N culture in 100 mL LB in 500 mL erlenmeyer.
2. Incubation at 250 rpm at 37°C until the DO 0,6 —> DO = 0.591
3. Cells were transferred to 3 sterile ice-cold 50 mL Falcon tubes. 20 mL in each falcon tube.
4. Incubate on ice for 30 min. Do not allow cells to warm up over 4°C.
5. Spin cells at 4000 rpm for 10 min at 4°C.
6. Discard supernatant and try to drain all remaining media.
7. Gently resuspend on 10 mL cold 0.1M CaCl2
8. Incubate on ice for 20 min
9. Centrifuge 10 min at 4,000 rpm at 4°C
10. Discard supernatant and gently resuspend on 5 mL cold 0.1M CaCl2/25% Glycerol
11. Transfer in 1.5 mL eppendorf (100 µL/tube)
12. Store at -80°C

NB: The competency of the prepared cells will be tested on the 20/09.