Notebook – Protocol – 8. Transformation

A. Purpose

Since the amount of DNA that comes with the DNA distribution kit is not enough for assembly, we need to transform this DNA into cells and then make our own stocks with sufficient amount of DNA. The principle of this step is to put the plasmid that contain a small amount DNA of interest into competent cells, let them grow (replicate) and then harvest back the amplified plasmid that contain the gene of interest.

B. Protocol

Transformation Method I

We used TSS method in this transformation process. The method created and developed by Chung, et al and the protocol was adapted in Dr. Arief Budi Witarto’s experiments.

Preparation

• Equipments : Centrifuge, horizontal shaker, incubator, micropipette, 1,5 ml microtube, 15 ml tube.
• Materials : LB culture for culturing the bacteria, E. coli BL21(DE3) competent cells as the cell host, plasmid Bba_J04450 50 pg/ul as the vector, TSS medium, 1 M glucose, TSS medium.

1. Pre-culture

• Culture the Escherichia coli BL21(DE3) competent cells by adding 100 µl of E. coli. BL21(DE3) glycerol stock into 3 ml of LB liquid medium.
• Incubate with shaking it at room temperature (28-20 °C) for 3 hours.

2. Plasmid transformation

• After 3 hours, add 1 ml of the pre-cultured results into 3 microtubes 1,5 ml steril.
• Pellets the cells by centrifugation at 4°C for 10 minutes.
• Discard as much as supernatant as possible.
• Mix the pellets in each tube by adding 1 ml TSS medium into the tubes. Suspend the pellets thoroughly with a micropipette. Suspended pellets then move into another tube that contain pellets, and suspend it again until we get 1 tube contain suspended E. coli.
• Move 100 µl suspended E. coli into 2 cooled microtubes. (One tube using for control and another one for the transformation reaction).
• Add 2 µl plasmid/part BBa_J04450 50 pg/µl from part registry into 100 µl suspended E. coli.
• Resuspency by pipetting and tapping the tube.
• Incubate the tubes for 5-6 hours at 4 °C.d E. coli.
• After 5-6 hours, add 1 ml LB liquid medium that has been mixed with 18 µl of glucose 1 M.
• Incubate overnight with shaking in room temperature (28-30 °C).

3. Plate out the cultured

• Pour 100 µl of the transformation results to agar plate containing appropriate antibiotic. We use CAM with 50 µg/µl final concentration in plate.
• Use spreader to spread evenly then incubate overnight at 37 °C .
• Centrifuge the remaining transformation results (900 µl) for 3 minutes
• Move 700 µl supernatant into empty tube.
• Pipetting the remaining supernatant with pellets (200 µl)
• Add 100 µl to LB plate containing appropriate antibiotic

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