DNA Transformation

I did the Transformation of E. cloni 10G Chemically Competent cells:

1. Remove Recovery Medium from the freezer and bring to room temperature. 

2. Remove E. cloni 10G cells from the -80°C freezer and thaw completely on wet ice (10-15 minutes). 

Tube (+)

Tube (D)

Tube (I)

3. Thaw the tube of pRham™ Vector DNA and microcentrifuge the tube briefly
to collect the solution in the bottom of the tube. 

4. Add 2 µL (25 ng) of the pRham Vector DNA and 1 to 3 µL (25 to 100 ng) of insert
product to the cells. Stir briefly with pipet tip; do not pipet up and down to mix, which can
introduce air bubbles and warm the cells. 

5. Important: Transfer the mixture of cells and DNA to a pre-chilled disposable polypropylene 15- mL
culture tube (17 x 100 mm). Performing the heat shock in the small tube in which the cells are
provided will significantly reduce the transformation efficiency. 

Summary: 

• Put 2 µL Prham ™ C-His + 3 µL transformation to tube (+)
• Put 2 µL Prham ™ C-His + 3 µL (DDro 1.5)  to tube (D)
• Put 2 µL Prham ™ C-His + 3 µL (mmA 1.5)  to tube (I)

6. Incubate culture tube containing cells and DNA on ice for 30 minutes. 

7. Heat shock cells by placing the tube in a 42oC water bath for 45 seconds. 

8. Return the tube of cells to ice for 2 minutes. 

9. Add 960 µL of room temperature Recovery Medium to the cells in the culture tube. 

10. Place the tube in a shaking incubator at 250 rpm for 1 hour at 37°C. 

11. Plate 100 µL of transformed cells on LB (or YT) agar plates containing 30 µg/mL kanamycin.

12. Incubate the plates overnight at 37°C. 

Transformed clones can be grown in LB, TB, or any other rich culture medium for preparation
of plasmid DNA. Growth in TB medium gives the highest culture density and plasmid yield.
Use kanamycin (30 µg/mL) to maintain selection for transformants.
Glucose may be added to 0.5% final concentration to ensure complete lack of expression
of the recombinant plasmid.