Competent Cell Preparation and Bacterial Transformation

1.  You will be given a culture tube containing containing 4 ml of E-coli bacterial cells that were grown to an O.D. 600 of between 0.3 to 0.5. Come to the big centrifuge at the front of the lab room. We will spin the tubes down at 2500 rpm for 10 minutes to pellet the cells.

2. Decant the growth medium into the waste bottle carefully. Remove all excess liquid from the cell pellet using a sterile p-200 tip. We must work sterilely today.

3.  Add 0.1 ml (100 microliters) of ice-cold 50mM Calcium Chloride (CaCl2) to the pellet and resuspend the pellet.

4.  Once the pellet is resuspended, add an additional 0.8 ml of the 50 mM CaCl2, invert to mix, and put the tube on ice for 30 minutres.

5.  Spin the tubes in the big centrifuge for 5 minutes at 2500 rpm. Remove the CaCl2.

6. Resuspend the competent cells in 0.25 ml (250 microliters) of 50 mM CaCl2 by flicking the bottom of the tube. Try to avoid the vortex unless your pellet just will not resuspend. If you need the vortex, use it on lower speed.

7.  Put 200 microliters of your competent cells into a clean microfuge tube on ice. Keep the remaining competent cells on ice also to be used later.

8. Get one of your DNA preps from last week. You will be told which to use for today.  Thaw the DNA quickly in your hand. In a sterile microfuge tube, put 4 microliters of TE. Then add one microliter of your DNA to that. Put the remainder of your DNA back at -20oC with your other unused DNA sample.

9. Briefly vortex the DNA plus TE, zip spin down. Add 3 microliters of this to the 200 microliters of  competent cells you made in step 7. Gently mix with a pipette tip up and down a few times. Do not vortex!

10.  Incubate on ice for 30 minutes.

11.  Transfer tube to the 42oC water bath for exactly two minutes. Cool for 30 seconds on ice.

12. Add all your transformed cells to the 1 ml of LB in the other 15 ml tube.

13.  Bring tube to the shaker at the front of the lab room. We will shake the tube at 37oC for 45 minutes. This allows the ampicillin resistance gene on the plasmid to be expressed before plating the cells onto plates that contain ampicillin.

14.  You will be given 2 sterile cell spreaders. You will also be given 4 LB plus ampicillin agar plates. Onto these plates you will be adding either 100 microliters of your culture, 200 microliters of your culture, or 500 microliters of your culture for spreading. One plate will be spread with the 50 microliters of untransformed competent cells that you saved in step 7.

15. To plate your cells: Go to the shaker and remove your LB cultures and put the tube at room temperature. Have your sterile spreaders ready (be sure to avoid touching the spreader flat surface that will be touching your cells. Dot the liquid around on the agar surface. Open the lid of an LB plus amp plate marked 100 microliters and dot 100 micoliters of your transformed cell culture onto the surface of the plate taking care not to touch the pipet tip to the surface of the plate. Then immediately take the sterile spreader and evenly spread the cells over the agar surface without gouging into the agar. Return the cover to the plate. Repeat this procedure adding 200 microliters to a new plate marked 200 and then again to by adding 500 microliters to another new plate marked 500.. For the transformed cultures, plate the 100 microliters first, then 200, then 500 using the same spreader. To a fourth plate, using a new spreader, plate the 50 microliters of competent cells you put aside in step 7. Dispose of your spreaders in the biohazard bag.

16.  Invert your plates (be sure they are correctly labeled on the bottoms. Include your 411 section number and your group number). We will place them into a 37oC incubator overnight.

17.  At least one group member will need to return between 2 and 4 tomorrow to check the results of the transformation and to count colonies. Retrieve your plates from the incubator. Llook under each plate for the presence of  colonies, indicating a successfully transformed bacterial cell. Do not remove the cover of the plate. Count the number of colonies you see on each of your plates, including the control plate that received only the competent cells (no DNA added). Hopefully, there will not be any colonies on the control plate. You may see colonies only on the 500 plate or, if your transformation was particularly successful, on the 200 or even 100 plates also. Count all plates that have colonies. Share these results with all of your group members. Place the entire stack of plates into back into the incubator. At 4 p.m. I will put them into the cold room until we use them next week.