Competent Cell Preparation

Prepare TSS (Transfection Storage Solution)

4.5 ml 50 ml

10% Polyethylene glycol 0.5g (Solid) 5 g

5% Dimethyl Sulfoxide 0.25 ml (Pure DMSO) 2.5 ml

20mM MgCl2 0.1 ml 1M 1 ml

Bring to 4.5 ml with autoclaved LB

Filter through 0.22 μM syringe filter.

Make and autoclave 35ml LB for each competent cell preparation.

Autoclave centrifuge tubes, flasks and bottle/beaker for TSS.

Place TSS and labeled microcentrifuge tubes on ice (12-30 tubes/prep)

If starting from store bought cells:

1. Inoculate 5 ml LB (w/o antibiotic) from company stock stored in –80C and let grow o/n.

2. Inoculate 30 ml LB (w/o antibiotic) with 300 μL from the o/n culture.

(Alternative: Inoculate 3 ml for 5 hrs and dump into 30ml culture.)

3. Monitor OD until it reaches 0.4-0.5.

4. When OD reaches 0.4 remove from shaker and put on ice.

5. Pellet the cells in 50ml falcon (or autoclaved tube) tube at 6000g, 4C for 10

minutes.

6. Decant liquid and place cells back on ice.

7. Resuspend cells in 3.0ml TSS, pipet up and down and vortex. (resuspend in 10% original volume of TSS. ie. 30ml culture, 3 ml TSS)

8. Pipet 250 μL cells into each prelabeled and precooled microfuge tube

9. Store at –80C

(Note: If make a pLysS or E strain use 34g/L chloroamphenicol in all media)