热激转化(DH10B)

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.