Prior to getting cells:

1) Turn on 42 deg bath.  Takes about 30 min to reach 42 deg.

2) Put 0.1 M sterile CaCl₂ on ice.

3) One tube of cells is good for several transformations.

For two transformations:

1) Put 10 μl of your ligation in the bottom of a 2059 Falcon tube. Place on ice.  For more than 2 transformations you may to use less than 10 μl per tranformation to not go over 20%.

2) Take one tube of cells out of  -80 ℃ and place on ice.  Defrost the cells by adding 100 μl of ice cold CaCl₂ and pipette gently up and down.  Total volume will be approximately 130 μl.

3) Add 50-60 μl of resuspend cells to each ligation.  Tap gently to mix.

4) Leave on ice for 15 min.

5) Heat shock for 90 sec at 42 ℃.

6) Add 1 ml pre-warmed or RT LB.

7) Put tubes in 37 ℃ shaker for 1 hr.

8) Pipette cells into eppendorf.

9) Spin down at 4000 rpm for 1 min.

10) Remove 800 μl of supernatant.

11) Resuspend remaining cells by gently pipetteing.

12) Plate 50 μl  and 150 μl on a pre-warmed plate with appropriate selection.

13) Leave O/N at 37 ℃ and think happy thoughts.