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TSS (Transformation Storage Solution)

Used to make TSS Competent Cells and Transformation. Because DMSO reacts with the syringe filters we use, you must first make 1.05× concentrated (50/47.5) base, filter sterilize, then add 2.5 mL of DMSO to 47.5 mL of the concentrated base. From OpenWetWare

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• 5.26 g PEG 8000
• 1.579 mL 1M MgCl₂ (or 0.316g MgCl₂·6H₂0)
• Add LB to 50 mL
• Filter sterilize (0.22 μm filter)
• Transfer 47.5 mL to a sterile tube
• Add 2.5 mL DMSO.
• Store at 4°C

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Competent Cell Preparation

Adapted from OpenWetWare

1. Grow a 5 mL overnight culture of cells in LB media. In the morning, dilute this culture back into 25–50 mL of fresh LB media in a 200 mL conical flask. You should aim to dilute the overnight culture by at least 1/100.
2. Grow the diluted culture to an OD₆₀₀ of 0.2–0.5. (You will get a very small pellet if you grow 25 mL to OD₆₀₀ 0.2)
3. Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X mL, you will need X tubes. At this point you should also make sure that your TSS is being chilled (it should be stored at 4°C but if you have just made it fresh then put it in an ice bath).
4. Split the culture into two 50 mL falcon tubes and incubate on ice for 10 min.
   All subsequent steps should be carried out at 4°C and the cells should be kept on ice wherever possible
5. Centrifuge for 10 minutes at 5000xg and 4°4°C.
6. Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.
7. Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume that you spun down. You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
8. Add 100 μL aliquots to chilled eppendorfs and store at −80°C.80°C, or use immediately in transformations.

Transformation using KCM solution

From the Rauscher Lab at Duke.

5× KCM solution: 

• 0.5 M KCl
• 0.15 M CaCl₂
• 0.25 M MgCl₂
  Filter sterilize through 0.2 μM filter. Store at -20°C

Transformation procedure

• 100 μL cells
• 20 µL 5× KCM
• X µL DNA
• 80–X µL nuclease-free H₂O

1. Add above to a chilled 14 mL polypropylene tube
2. Keep on ice for 30 min
3. Heat shock at 42°C for 45 sec
4. Place on ice for 2 min
5. Add 250 µL of LB or SOC to each tube
6. Incubate at 37°C with shaking for 1 hr
7. Plate 250 µL of mix on LB with selective antibiotics
8. Incubate plates overnight at 37°C