Transformation Using Heat Shock

Transformation Protocol Using Heat Shock

1.)   Take competent E. Coli cells from the -80°C.

a. Use DH5α cells for most cases.

b. If cutting with XbaI or other DAM – enzyme site, use SCS110 cells which are deficient in DAM and DCM methylases.

c. If cloning with intent of hairpin formation use SURE cells.

2.)   Place competent cells in an ice bath to thaw.

3.)   Turn the 42°C water bath on.

4.)   For transforming a DNA construct, use 50 uL of competent cells. For transforming a ligation use 100 uL of cells depending on how competent they are (typically 50 uL of DH5α or SURE cells will be sufficient.)

5.)   Place the needed amount of cells into a 14 ml Falcon tube. Follow that addition with the addition of the total volume of ligation.

6.)   Cap and place in the 42°C water bath for 45 seconds.

7.)   Put the rube back on ice for 2 minutes to reduce damage to E. Coli cells.

8.)   Add 1 mL of LB (with no antibiotic). Incubate for 1 hour in 37°C shaker.

9.)   After incubation period is over, remove 900 uL and place into Eppendorf tube. Pellet by centrifugation at full speed for 1 minute.

10.) Remove 800 uL of LB and resuspend in the 100 uL of LB remaining.

11.) Plate the 100 uL that was left in the falcon tube and the concentrated 100 uL that is in the Eppendorf tube onto LB + appropriate antibiotic plates. Grow for 12 – 16 hours.

12.) Pick colonies and continue with mini prep.