• One week (5 lab periods of 45-55 minutes).

• The students think they have different DNA and the same enzymes -- but in fact you give them the same DNA (lambda) and different enzymes. Ultimately,
  
  Tube 1 has lambda DNA, and EcoR I enzyme. It represents "suspect 1."
  
  Tube 2 has lambda DNA, and BamH enzyme. It represents "suspect 2."
  
  Tube 3 has lambda DNA, and Hind III enzyme. It represents "suspect 3."
  
  Tube CS has lambda DNA, and Hind III enzyme. It represents DNA from the "crime scene."
  
  
  Tube U has lambda DNA, and distilled water. It represents control DNA from the "crime scene" that will remain uncut.
  
  
• Create a scenario, or use one of ours!
  
  

1. Electrophoresis buffer to put in the gel boxes. (Re-use it!)
   
   We use TAE buffer. In the gel boxes it is used at 1X, but it can be prepared in concentrated form (50X). Dilute with dH₂0 to give 1X concentration. You'll need 5-10 L of 1X TAE. TBE buffer may also be used (it can be made concentrated 5-10X).
   
   
2. 0.8 % agarose solution.             Makes 500 mL
   
   Mix 4 g of agarose with 1X TAE (your electrophoresis buffer), giving a final volume of 500 mL. About 5 min. of microwaving will dissolve the agarose. 500 mL is enough for one class. Hold the melted agarose in a water bath at 65°C; students dispense just what they need (25-30 mL/gel) and cast their gels when their agarose container is just cool enough to hold (about 50°C).
   
   
3. Restriction enzymes & enzyme buffers
   
   We use Hin d III, Bam H I, and Eco R I. Store enzymes in the freezer (they are mixed in glycerol so they never freeze). NEVER dilute enzymes unless you use the diluents provided by the suppliers! We have been successful diluting enzymes 1:3 up to 3 weeks prior to use. You'll use about 2 µL of each of two enzymes, and 4 µL of the third enzyme (CS and one suspect) per team. We use the same compromise ("generic") buffer, 2X RB, for all the enzyme digests.
   
   Remember, the way this lab is set up (as written for the students), what they think is "enzyme" is really lambda DNA, and what they think are different DNA samples, are really the three different restriction enzymes. The exception is with the "U" tube. The DNA for U is really uncut lambda DNA. It is sufficient to only have 2 µL of the uncut lambda to run on a gel.
   
   Sometimes DNA cut with EcoR1 has more than the number of bands expected. This is a consequence of less than optimal reaction conditions, especially low salt or high glycerol concentrations. The additional activity, known as EcoR1* (star) activity, recognizes a slightly altered 4-6 base pair sequence where it cuts the DNA.
   
   
4. Lambda DNA.
   
   We use uncut lambda, diluted with TE (10 mM Tris, 1 mM EDTA, pH 8) to a concentration of 0.1 µg/µL.
   
   NOTE: if you were to purchase uncut lambda DNA from a vendor, university lab or biotech company, it would be shipped to you in some other concentration -- for example, the label might read "500 µg / 971 µL." Convert this to a decimal: 0.52 µg/µL. This lab requires that the lambda be at the concentration of 0.1 µg/µL. Therefore, you would have to dilute it. You'll need about 20 µL of the lambda at the right concentration per team. For example, for a class of 24 teams you'll need about 500 µL of lambda DNA at a concentration of 0.1 µg/µL:

   	YOU WANT	YOU USE
   Set up a proportion:	(500 µL) (0.1 µg/µL)  =	( x ) (0.52 µg/µL)

   
   
   Solving for x: x = 96 µL. This means you put 96 µL of your concentrated lambda DNA in a clean tube, add 404 µL of TE, and mix. Voila! 500 µL of dilute lambda!
   

• This lab is intense. It assumes the students know how to use micropipets. Be prepared to demonstrate centrifuge use, electrophoresis, and gel staining and photography! Careful attention to detail is required.
  
  
• We recommend reading pp. 247-275 in DNA Science, A First Course in Recombinant DNA Technology, D. Micklos and G. Freyer, 1990, Carolina Biological Supply Company.
  
  
• Because human DNA restricted with an enzyme will give a smear and not defined bands on an agarose minigel, we recommend you do a follow-up activity to demonstrate a true RFLP analysis. Gene Connection modified an activity from a science teacher's journal of about 6 years ago. The concepts involved are:
  • restriction of human DNA
  • separation of fragments by size by agarose gel electrophoresis
  • transfer of DNA to a hybridizing membrane
  • conversion of double strand DNA to single strand DNA
  • exposiure of membrane bound DNA to a single stand radioactive "probe" of known sequence
  • binding of probes to specific complementary sequences, thus "tagging" the restriction fragments containing the sites of interest
  • exposure of radioactively probed membrane to photographic film. The radioactivity will expose the film, resulting in black bands on the film, corresponding to the "tagged" restriction fragments.
  • analysis of film for bands confirming/denying identity or relatedness.

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