Protocol for Transforming Competent E. coli Cells with Plasmid DNA

Written by Super User. Posted in DNA

Protocol for transforming competent E.coli cells with Plasmid DNA.

E.Coli Transformation Method

E.Coli Transformation Method - Making Competent E. coli Cells with Plasmid DNA

 

         Use competent E. coli cells frozen cells purchased from Gibco-BRL. Aliquoted as 50 uL aliquots in 1.5 ml microfuge tubes and stored in the -80 C freezer until needed.
Use 25uL save half.

When handling frozen competent cells is KEEP THEM COOL!

         They are very sensitive to being warmed and will not work as well if not kept on ice as much as possible.

         1. Obtain frozen cells from the -80 C and place them on ice immediately.

         2. Warm the tube with your fingers until the cells just begin to thaw and replace on ice.

         3. You do not need very much plasmid to get transformed colonies. Generally around 20 ng (nano grams) is enough.   (using 100ng of stock DNA; dilute 1:in H20 100ul).Take 1uL for transformation. (Add 5 ul or less plasmid DNA).

         4. Mix cells by briefly by finger flicking gently tube then incubate on ice for 5-30 min.

         5. Heat shock the cells by placing the tube in a 37 C water bath for 45 seconds and return to ice.

         6. After 2-3 min, add 1 mL of LB broth, place in shaker,and incubate at 37 C for 1 hr.

         7. After incubation, shake tube and aliquot 100 ul of the suspension into the middle of an LB/ampicillin plate as a puddle.

         8. Spread by evenly spreading cells over the plate using a bacterial spreader.Make sure you dip your spreader in ethanol and flame it
         before spreading. Flaming without ethanol seems to work ok also.

         9. Centrifuge the remaining cells in the microfuge for 30 seconds on low-medium speed, remove, and pour out most of the broth above the pellet of cells.

[Note: You will want to keep the rest of the bacterial cells so you never have to do this again. Add glycerol to the bacterial cultures, at about 50% by volume. Store this in the -80C freezer. This stock will hold for years. You can then thaw and use about 5 uL of glycerol stock bacterial per 5 mL of LB media. This takes 5 minutes but saves you hours of time in the future.]

         10. Resuspend the cell pellet in the remaining broth using a 200 uL pipetteman.

         11. Transfer the resuspended cells to the middle of another LB/ampicillin plate and spread as before.

         12. Place in the 37 C incubator with the agar side up and the lid side down. Incubate overnight, and check to see if colonies have formed the next day. Remove and store at 4C in a fridge that day. Wrap bacterial plates with plastic wrap to keep the moisture out, and store bacterial plates with the lid side down.