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.