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Transformation is the process of inserting foreign DNA into a cell in order to change (transform) the cell. We will be transforming our circular DNA plasmid from the Gibson Assembly reaction into special E. coli cells called XL1-Blue cells. These cells are special because they have been made “competent,” which means that they are readily able to take up our foreign DNA and produce large amounts of high-quality plasmid DNA. The plasmid will replicate inside the cells, and the bacteria will multiply, allowing us to purify many copies from a culture of the E. coli.
- Chemically competent XL1-Blue cells
- pET28b-GFP plasmid from Gibson Assembly
- Liquid LB media
- LB+Kanamycin plates
- Glass beads
Note: XL1-Blue and all chemically competent cells have been chemically treated using salt such calcium chloride to make the cells competent to take up DNA. As a result, the cells are very fragile and die easily. Keep the cells on ice at all times before the heat shock and try not to pipette the cells too vigorously. There are many different competent cells made for different purposes such as making high-quality plasmid DNA or expressing recombinant proteins.
- Divide your competent cells into two aliquots of ~50 µL, each in a 1.5 mL tube.
- To one aliquot, add 10 μL of your Gibson reaction to the competent cells. Ensure that you pipette your Gibson reaction INTO the liquid the cells are suspended in!
- To the other aliquot of competent cells, add an equivalent mass of the digested vector as a negative control.
- If you put 50 ng of vector into a 20 µL Gibson reaction and transformed 10 µL, the mass of vector you will transform is _____ ?
- Using the concentration of your digested vector, calculate the volume you need to add to transform an equivalent mass of your digested vector in the negative control transformation. This volume is _____ ?
- Hint: volume digested vector (uL) = mass transformed vector (ng) / concentration digested vector (ng/uL)
- Incubate cells on ice for 10 minutes.
- Heat shock the cells by placing the cells at 42°C for 45 seconds.
- Recover the cells on ice for 2 minutes.
- Add 450 µL of LB without antibiotics to each tube and incubate while shaking at 37°C for 60 min.
- Pellet cells by centrifugation in a microfuge for 1 minute at 13,000 rpm.
- Remove 300 µL of supernatant from each tube and resuspend the pellet in the remaining 200 µL LB.
- Spread cells on LB+Kanamycin plates using glass beads, leave to dry for ~10 minutes.
- Invert the plate and discard the glass beads in an appropriate waste container.
- Incubate the plates agar side up at 37°C overnight.