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TSS (Transformation Storage Solution)

Used to make TSS Competent Cells. 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

To make 50 mL:

• 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

Transformation

Adapted from OpenWetWare

1. Grow a 5 mL overnight

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1. culture of cells in LB media. In the morning, dilute this culture back into

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1. 25–50 mL of fresh LB media in a

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1. 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.

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1. 2–0.5. (You will get a very small pellet if you grow

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1. 25 mL to OD₆₀₀ 0.2)
2. Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X

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1. 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

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1. °C but if you have just made it fresh then put it in an ice bath).
2. Split the culture into two

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1. 50 mL falcon tubes and incubate on ice for 10 min.
   All subsequent steps should be carried out at 4

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1. °C and the cells should be kept on ice wherever possible
2. Centrifuge for 10 minutes at 5000xg and 4

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1. °C.
2. 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.
3. 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.
4. Add 100

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1. μL aliquots to chilled eppendorfs and store

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1. at −80

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1. °C.

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