Bacterial Cell Transformation

Materials Needed: 50 uL competent cells, DNA to transform (from Gibson or otherwise), ice, 42 deg water bath, 2 agarose plates w/ proper selection media, 500 uL SOC media, plate spreader, ethanol, bunsen burner.

  * Make sure agarose plate contains the same resistant as the DNA being transformed.
 * 1) Fill ice bucket, take to -80 deg freezer. Take out tube of competent cells ***For Gibson Transformations: Use the XL10 Gold cells in box labeled 50 (on bottom layer of left-most rack, second from top shelf). They are aliquoted in 50 uL aliquotes.                                                 *For transformations requiring BL21 cells: cells are in box labeled 60 (on left-most rack, second from top shelf) See Protein Expression in BL21 Cells for the correct protocol.
 * 2) If necessary, aliquot 50 uL of cells into fresh 1.5 mL tube. **Mark in-use tube of cells with a dot on lid.
 * 3) Add half of Gibson reaction mixture to competent cells.
 * 4) Incubate on ice for 15-20 minutes. In the meantime: Refreeze rest of competent cells in -80. Fill up appropriate # wells in 42 deg heat block with water.
 * 5) Heat shock cells in water-filled 42 deg heat block for 45 seconds.
 * 6) Place back on ice for 2 minutes.
 * 7) Add 500 uL SOC media **Careful not to tip bottle or get any media in cap! And open next to a flame!
 * 8) Shake tubes for 1 hours @ 37 deg C.
 * 9) Grab the agarose plates from the refrigerator and place them in the incubator.
 * 1) If transforming a ligation or Gibson reaction where efficiency may be low, plate cells on 2 plates as follows: 50 uL onto one plate and rest of cells onto second plate. Spread w/spreader. If transforming a plasmid where efficiency should be high, plate 5-20uL. Place in incubator overnight.
 * 2) Next morning, check plates. Remove plates from incubator when size of colonies is a little bigger than the tip of a pipet tip. Store in 4 deg fridge (parafilm for longer storage) if not using immediately.