Introduction
By mixing E. coli cells with plasmid DNA carrying an antibiotic resistance gene, one can isolate cells that have become transformed with the foreign DNA but the frequency of this event is so vanishingly small that it is not feasible. However, E. coli cells can be made "competent" to take up foreign DNA by treating them with CaCl2 on ice. It is thought that the Ca++ ions form an ionic bridge between the negatively charged Phosphate groups of both the DNA and the membrane phospholipids. The cold temperature crystallizes the membrane stabilizing this interaction. When the cells are then exposed to a "heat shock" some of the cells take up the DNA. To make cells highly competent one starts with a culture in the log phase of growth. If at any time during the procedure the cells are allowed to warm up above 4 deg C they will not become competent. Be sure to use good sterile technique during the entire procedure. Transformation efficiency can be measured by counting the number of transformants obtained per ug of DNA.
Procedure for Making Competent Cells
1. You will be provided with a flask of ice cold E. coli cells harvested in mid log phase growth. Transfer these cells to two ice-cold 15 ml centrifuge tubes and pellet the cells for 10 min at 4000 rpm at 4 deg C. Be sure to balance the tubes.
2. Pour off the supernatant into a waste beaker. Be careful not to disturb the pellet.
3. Using a sterile pipet, add 5 ml of ice-cold 50 mM CaCl2 to the cells and swirl the tubes to resuspend the cells. Do NOT leave the tubes out of the ice bucket for prolonged periods of time. If necessary, resuspend the cells by pipetting into and out of a blue-tip pipetman several times.
4. Return the tubes to ice and incubate the cells for 20 min.
3. Centrifuge the cells a second time under the same conditions as in step 1.
4. Repeat step 2.
5. Add 0.5 ml of ice-cold 50 mM CaCl2 to each tube and resuspend the cells as in step 3. No white clumps of cells should be visible. Pool the cells into one tube and swirl to mix.
6. Transfer the resuspended cells into two sterile ice-cold 1.5 ml Eppendorf tubes. Keep one tube of competent cells on ice for the transformation procedure and store the second tube in the -80 deg C freezer.
Procedure for Transforming E. coli Cells
1. Place 3 sterile 15 ml tubes on ice and label all with your group name and either + (your plasmid name), No DNA, and Control DNA. (What are the two controls for?).
2. Add 5 uL of your ligation mix to one tube, 5 uL of water to another and 5 uL of pUC19 control DNA to the third.
3. Add 100 uL of competent cells to each tube (keep them on ice). Swirl to mix and incubate on ice for 20 min. While the cells are incubating, label 3 LB amp plates with the three transformation mixes as in step 1. Also label an LB (no amp) plate. Onto this last plate you will transfer cells from the control DNA transformation. Why?
4. Following the 20 min incubation, transfer all three tubes to a 42 deg C waterbath for EXACTLY 90 seconds. Then transfer the tubes back to ice for 1 additional minute.
5. Add 1 mL of LB broth to each tube and place in a shaker incubator at 37 deg C for 30 min. This allows the transformed cells to begin expressing the ampicillin resistance gene prior to being placed on medium containing ampicillin.
6. Transfer 200 uL of the final mix (transformed cells + LB) onto each appropriate plate and spread with an glass rod and spreader platform. Be sure to flame the ethanol-treated glass rod prior to EACH use.
7. After waiting 5-10 min for the liquid to be absorbed into the agar plate, incubate the plates upside down at 37 deg C over night.
8. Streak at least 4 resulting colonies on fresh LB amp plates to isolate transformed cells. Grow a single colony from each of the streaked plates in LB amp broth for minipreping and sequencing.