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STUDENT OBJECTIVE

Students spool DNA fibers from lysates of onion cells and colorimetrically demonstrate that the sample is DNA. Students perform a plasmid transformation of E. coli, demonstrating the acquisition of ampicillin resistance.

EQUIPMENT	AMOUNT
	(Class of 24 with 8 groups)
Blender Incubator, 37oC Water baths at 60o, 50o, 42oC, and 37oC (can be improvised) Ice Bath Spectrophotometer, 600 nm Centrifuge (3000 G) Balance, double pan for centrifuge tubes	1/lab 1/lab 3/lab 1/group 1/lab 1/lab
MATERIALS
DNA isolation: Large onion Solutions 100 ml of commercial Woolite in 900 ml of 1.5% NaCl Cold 95% ethanol Ice
DNA assay: Pipettes or dispensers, 5 ml in 0.1 ml Test tubes Rack Spectrophotometer cuvettes Aluminum foil Solutions 4% NaCl Diphenylamine (CBS#85-8330) Herring testes (Sigma#6898)* Acetaldehyde (to make reagent) Sulfuric acid (to make reagent) Glacial acetic acid (to make reagent)   Transformation procedure:	4/group 6/group 1/group 2/group 1 roll
Complete kit for six groups available (CBS#21-1142)* or order separately as below E. coli (plasmid free) culture (CBS#21-1530) or (CBS#21-1531) Petri plates (sterile) Luria agar (CBS#21-6620)* Luria agar + ampicillin (CBS#21-6621) Bacteriological metal transfer loop Alcohol or Bunsen burner Tubes, plastic (size important—9.5 cm by 1.5 cm) (CBS#21-5080) Pipettes, sterile disposable transfer (CBS#73-6895A)* Glass hockey sticks, sterile Solutions	1/lab 1/lab 4/group 1/group 1/group 2/group 6/group 4/group
CaCl2, 50 mM pAMP ampicillin resistance plasmid, 0.005 ug/ml (dilute CBS#21-1441) Luria broth (CBS#21-6660)* 70% ethanol 10% solution of household bleach Ampicillin (CBS#21-6860) if making all media from scratch
Autoclave bags for wastes Ice in 500 ml beaker	1/group 1/group

*Please refer to the Appendix for name and address of supplier.

PREPARATION

Four Weeks before Lab

Considerable time can be saved by ordering colony transformation kits or premade media and agars from CBS. These should arrive one week before lab. If this is not done, place orders for separate items with suppliers to arrive two weeks before lab.

Two Weeks before Lab

1. 1.5% NaCl preparation

   15 g NaCl/1000 ml water

2. 4% NaCl preparation:

   20 g NaCl/480 ml water

3. Diphenylamine preparation:

   This can be ordered as premade solution from CBS or made from crystals. Adjust volumes and amounts as necessary.

   Caution: Strong acids. Wear goggles and work in fume hood. Dissolve 15 g diphenylamine in 1 L fresh glacial acetic acid. Add 15 ml concentrated sulfuric acid. Store in dark bottle. On day of use, add 1 ml acetaldehyde solution (1 ml of acetaldehyde in 500 ml water) per 100 ml of diphenylamine solution. Lab safety is improved if this solution is dispensed by students from an automatic pipetter.

4. DNA standard solution preparation: 500 ug/ ml
   Dissolve 50 mg DNA in 100 ml 4% NaCl; total volume as needed.

   Can be stored frozen.

5. 10% household bleach preparation:
   Add 100 ml bleach to 900 ml water.

One Week before Lab

1. If premade agar was not ordered, make luria agar following supplier’s directions. (Can order as dehydrated media, CBS#21-6700 or as a premade agar, CBS#21-6620.*) After autoclaving, but before gelling, split medium in half and add ampicillin to one-half. Pour into sterile plastic petri plates to a depth of approximately 5mm. Each student station should have four plates: two with and two without ampicillin.
2. If premade luria broth was not ordered, make Luria broth following supplier’s directions. (Can be ordered as dehydrated media, CBS#21-6710.*) Package 2 ml in an autoclaved small vial. Each student station should have one vial.
3. If premade plasmid, pAMP (CBS#21-1441)*, solution was not ordered, prepare plasmid solution. Sigma also sells a number of plasmids; pBR322 confers ampicillin resistance and can be ordered as a concentrated frozen solution (#D4904)* containing approximately 50 ug of plasmid DNA. Dilute to 0.005 ug of plasmid DNA per ml with sterile 50 mM CaCl₂ solution or TE Buffer. (See #4.) Package 0.1 ml per small sterile vial. Each station should have one vial.
4. 50 mM CaCl₂ solution preparation:
   5.6 g CaCl₂/1000 ml distilled water

Two Days before Lab

Streak plasmid-free E. coli on the surface of a luria agar plate and incubate at 37°C for 48 hours. Students will scrape cells off this plate and use them in the transformation procedure. One plate per lab section is ample

Day before Lab

1. Put several pint bottles of 95% ethanol in a freezer so that it is cold for DNA isolation.

NOTES

1. DNA isolation can be done as a combination demonstration/student activity. Instructor can dispense DNA-containing solution to students. They can add the cold 95% ethanol and collect the DNA fibers. Students can perform colorimetric test as described or instructor can collect DNA from students, dissolve it, and run colorimetric test as a qualitative one using student DNA, known DNA, and solvent blank. The blue color demonstrates that they have isolated DNA.
2. Transformation procedure works very well when students pay attention to details. Demonstrate aseptic technique to students before they start (how to hold hands, lids, and pipettes; flaming loops, etc.). Because of the exceedingly small volumes involved, students must be very careful in adding reagents to plastic tubes. If they touch pipettes or loops to the side, the drip often clings and does not mix with the other reagents. Also, the temperature and duration of the heat shock are critical, as is the size of the plastic tube. The treatment must be exactly 42^oC for 90 seconds with the cells coming from the ice bath to the high temperature back to the ice bath immediately. After two minutes in the ice bath, we have obtained good results by adding 250 µl of Luria broth and incubating the tubes in a 37^oC water bath for 30 minutes to allow a recovery time. If low numbers of transformed cells are obtained, double the amount of plasmid used. You should obtain about 25—50 colonies of transformants on the ampicillin-containing plate.
3. CBS has an excellent booklet describing the colony transformation experiment in detail. It is supplied with the colony transformation kit.
4. Some strains of E. coli can cause gastrointestinal distress. Glassware should be washed immediately. Disposables should be collected in autoclave bag and autoclaved before disposing. Students should wash their hands after any contact with cultures.

CLASSROOM SUGGESTIONS

1. If time is short, the colorimetric test for DNA can be run as a demonstration. Do not use the standard curve and treat the test as a qualitative one with a blue color indicating the presence of DNA. Collect samples from the students to run the test.
2. Check out the links for this lab topic at http://auth.mhhe.com/biosci/genbio/dolphin/ You will find useful materials for developing your lab introduction or summary, and in some cases, you may want to tell students to connect to a particular site for further information.

ANSWERS TO CRITICAL THINKING QUESTIONS

1. 1) Competent, plasmid-free E. coli was a suitable host in which to insert a foreign gene.

   2) Plasmid DNA carrying the ampicillin-resistance gene was the vector to carry genes to the host.

   3) Luria agar plus ampicillin was used to isolate those host cells that had taken up the plasmid.

2. Answers will vary but should include discussion of insertion of gene, vector, host cell, and selection method. Students will most likely discuss utilization of antibiotic resistance as marker gene and should detail complications of transferring antibiotic resistance to humans.
4. Possible sources of error include:
   — transference of agar with initial E. coli cell mass.
   — failure to take up a "loopful" of plasmid.
   — incorrect temperature of water bath.
   — incorrect timing of heat shock/ice bath procedure.
   — inaccurate pipetting technique.
   — spreading inoculum with too hot a "hockey" stick.
   — overincubation.
   Clerical errors such as mislabeling plates, inoculating onto wrong plate or recording data incorrectly.
5. The No plasmid, normal media plate "controlled" the media and E. coli viability.
   The No plasmid, ampicillin media "controlled" the ampicillin activity.
   The plasmid treated, normal media "controlled" the transformation procedure and E. coli viability.

SUPPLEMENTAL MATERIALS