Schedl Lab

Washington University Genetics

Heat shock transformation (DH10B homemade)

Heat Shock Transformations (DH10B)

Prepared by Ziva and adapted by Maia Dorsett

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 ul 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 ul per tranformation to not go over 20%.

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

3) Add 50-60 ul 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 C.

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

7) Put tubes in 37 C shaker for 1 hr.

8) Pipette cells into eppendorf.

9) Spin down at 4000 rpm for 1 min.

10) Remove 800 ul of supernatant.

11) Resuspend remaining cells by gently pipetteing.

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

12) Leave O/N at 37 C and think happy thoughts.

  • Tim Schedl, Ph.D
    Department of Genetics
    Campus Box #8232
    Washington University School of Medicine
    4566 Scott Ave.
    St. Louis, MO 63110

    Contact

    Email: ts@genetics.wustl.edu
    PHONE: (314)362-6164 [lab]
    FAX: (314)362-7855