18.2: Exercise

Exercise A

Procedure for bacterial transformation:

1. Using a micropipette and sterile tip, add 250 µL cold 0.05 M CaCl₂ into two microcentrifuge tubes (microtubes). Label both microtubes with your group name.
2. Obtain a plate of E. coli. Sterilize an inoculation loop by flaming it from the base to the tip slowly and then cooling it by placing the loop gently onto the agar plate where no bacteria are growing. Transfer a large colony of E. coli to one of the microtubes. Be careful not to pick up any agar.
3. Place your loop into the CaCl₂ solution and mix well to dislodge the cells into the solution.
4. Immediately suspend the cells by pipetting up and down 10 times using a 100 µL micropipette with sterile tip. Mark this first tube “+” and return it to the ice.
5. Repeat steps 2 to 5 for the second tube. Mark this tube “-“. Sterilize your loop again once you are finished. Make sure your loop has cooled before the next step.
6. Using a micropipette and sterile tip, transfer 10 µL of pAMP plasmid directly into the cell suspension in tube “+” only. Be sure the volume on your micropipette is set correctly. Then, immerse the tip into the “+” solution and pipette up and down several times. Make sure your micropipette tip is placed directly into the solution and does not touch the wall of the microtube. Mix well. Return the tube to ice for 15 minutes.
7. While the tubes are incubating on ice, obtain two Luria Broth (LB) agar and two LB/Amp agar (Luria Broth containing ampicillin) plates. Label one LB agar plate “LB+” and the other “LB-“. Label one LB/Amp plate “LB/Amp+” and the other “LB/Amp-“. Label all plates with your group name, E.coli, the date, and 37°C. All labels should be written on the base of the plates, not the lids.
8. Heat-shock the cells in both the “+” and “-” microtubes by transferring the tubes rapidly from ice into the 42°C water bath for 90 seconds. This transfer must be rapid for a sharp and distinct shock, which optimizes transformation of the pAMP plasmid into E. coli. Immediately return the cells to ice for 2 minutes.
9. Use a micropipette and sterile tip to add 250 µL cold Luria broth to each tube. Mix well and leave the tubes at room temperature for recovery for 10 minutes. During this time, the Amp^R gene that has been newly introduced into E. coli is expressed. As a result, β-lactamase, an enzyme that cleaves the functional group used by some antibiotics to kill bacteria, is produced. The transformed cells are now resistant to ampicillin since they contain the gene whose product renders the antibiotic ineffective.
10. Place 100 µL of the “-“ cells onto the “LB-” plate and 100 µL of the “-” cells onto the “LB/Amp-” plate. Place 100 µL of “+” cells onto the “LB+” plate and 100 µL of the “+” cells onto the “LB/Amp+” plate.
11. Immediately spread the cells using a sterile spreading rod. Immerse and remove the spreading rod from ethanol and briefly pass it through a flame. (Do NOT flame sterilize if you are using plastic single use spreaders.) Cool the rod by gently touching it to the agar on a region of the plate away from the bacteria. Spread the cells throughout the plate. Sterilize the rod again before you spread the “+” cells and once more when you are finished spreading the cells. Allow the plates to set for 5 minutes right-side up (lid up). Then, tape your plates together and incubate upside-down (lid down) at 37°C for 24-48 hours.

Exercise B

Procedure for making a recombinant plasmid model via restriction enzyme digest

Your instructor will provide a paper replica of the E. coli plasmid. The unshaded piece of DNA represents a circular E. coli plasmid. The shaded pieces of DNA represent a segment of DNA taken from a human chromosome. Restriction enzyme HindIII is isolated from Haemophilus influenzae and recognizes the following restriction site and produces fragments with “sticky ends”:

5’ A A G C T T 3’

3’ T T C G A A 5’

Cut and tape the unshaded strand together to form a circular plasmid of bacterial DNA.

Tape the shaded (human DNA) pieces together to form a long, linear, strand.

Locate the cut site(s) on the plasmid DNA and on the linear strand of DNA. Draw it in pencil as they would be cut by HindIII and then show them to your instructor for approval.

Once the instructor confirms that you have the correct cut sites, use a pair of scissors to cut the DNA of both DNA molecules. Note, your scissors represent the restriction enzyme HindIII.

Lastly, insert the isolated (target) human DNA into the plasmid and tape the fragments together. You now have a recombinant DNA plasmid.