Gila Stein                                                                                        Return to Biology Menu

Ramaz Upper School

Summer 2001

 

An Exercise in DNA Manipulation

Extraction and Digestion of Calf Thymus DNA

 

Purpose: To help students visualize that there is something called DNA and to allow them to manipulate the DNA using a contemporary laboratory technique. 

 

Materials:

Centrifuge tubes, centrifuge, gel box, blender, pipettes, pH meter, staining boxes, test tubes, methylene blue, calf thymus, required solutions

 

Solutions:

Thymus DNA Extraction - Prep Buffer

 57 g table sugar

 3.1 g Epsom salts

distilled H20/500 ml.

pH 7.5 adjusted with1 N HCl/NaOH (add one drop at a time)

6g Buffered aspirin (about 1 tablet)

     

SDS

liquid detergent and tap water to make 20% solution

 

NaCl

9.2 g table salt

distilled water to make 250 ml

95% Ethanol - ice cold(put in freezer night before in plastic bottle)

 

Restriction Enzyme Digestion:

Extracted DNA in volume of 20ul

10x buffer (16ul NEB Buffer/8ul NEB 2 buffer)

5 ul of EcoRI, HaeIII, NotI enzymes

35ul distilled water

Gel electrophoresis

250ml Methylene Blue stain

9 ul loading dye

Distilled water

 

Procedure:

Below is the procedure for groups of 4 students each. The entire process will take 4-6 days.

I. Isolate nuclei 

a. Mix 200 ml of prep buffer with 10 ml thymus. Puree in blender.

b. Strain using a piece of cheese cloth - save the liquid.

c. Centrifuge 4 ml. of strained liquid to make a pellet of nuclei.

d. Pour off liquid (supernatant) from top of pellet.

e. Resuspend pellet in 2 ml of prep buffer. Stir with plastic pipette.

f. Put 40 drops of resuspended pellet into a clean test tube for each lab group.

    

II. Lyse nuclei

a. Add 6 drops of SDS composite.

b. Add 5 drops of NaCl solution, one drop at a time, mixing gently after each drop.

 

III.  Precipitate DNA

a. Gently add 1ml of 95% iced Ethanol by pipetting down the side of the test tube. The alcohol will form an overlay.

b. Spool the DNA where the ethanol and the DNA mixture meet. The DNA  will look like gobs of white mucus. Wrap this substance around a glass pipette and lift out of the tube. Place into a centrifuge tube for later. Put in freezer.

 

IV. Purifying the DNA

a. Remove spooled DNA from freezer and thaw.

b. Centrifuge for 30 seconds. Pour off supernatum.

c. Add 1ml of pineapple juice (papaine). This will get rid of any foreign proteins on the DNA. Pour off after 2 minutes.

d. Add 1ml of 100% ethanol. This will get rid of the papaine. Pour off after 2 minutes.

e. Resuspend the DNA in TE for 30 minutes. Use same amount as the DNA in the tube. The DNA is going into solution to be aliquoted from the top of the tube later on. Put in freezer.

     

V. Preparing the Gel

 a. Prepare an agarose gel. This procedure will take about 30 minutes. Let gel set in TAE overnight.

 

VI. Preparing Restriction Enzyme Digests 

a. To prepare your digestion enzymes you will need to aliquot the proper amounts of each into a centrifuge tube.

 DNA                         20ul                          

10x buffer                 4ul                            

Enzyme                    4ul                            

H2O                         12ul                          

Total                         40ul                          

b. Label all of your tubes. You should complete 3 tubes. There will be enough digest in these 3 tubes for your group of four to separate into groups of two. Each group of two will load its own DNA, although the group of four will share a gel.

c.      Place your centrifuge tubes in 370C for 30 minutes where they will digest (cut).

 VII.    Loading Samples Into The Gel Wells

 a. You will load your samples into the wells in the following order: Load 20ul sample and 3ul loading dye in wells # 2 - 7.

well #1: 123kb (load 10ul) well                    well #5: HaeIII(20ul) Group Ib

well #2: EcoRI (20ul) Group Ia                     well #6: NotI (20ul)   Group Ia

well #3: EcoRI (20ul) Group Ib                     well #7: NotI  (20ul)  Group Ib

well #4: HaeIII (20ul) Group Ia                      well #8: 1kb (load 10ul)

b. When all samples are loaded, close the lid on the gel box and attach the electrical leads. The black lead attaches to the side of the gel with the wells. The red attaches to the opposite side of the wells. Run the gel for approximately 45 minutes.

VIII.      Staining The DNA Bands On The Gel

a. USE GLOVES WHEN WORKING WITH METHYLENE BLUE STAIN

Pour liquid out of gel box. Carefully slide gel into staining tray. Pour  Methylene Blue stain into tray (not directly onto gel) to just cover the gel. Let stain for three hours. Pour off stain into sink - flush sink with running water. Add 250ml of distilled water to staining tray. Destain for 12-18 hours.(This time can be cut back if methylene blue is diluted.) Agitating on an orbital agitator will speed up destaining. Remove gel from the tray and wrap in plastic wrap, then place in the refrigerator.

 

IX. Comparison Of DNA Cuts Using Different Restriction Enzymes

 a. Place your gel on the overhead projector to view your DNA fragments

 showing the cuts which were made by each enzyme.

b. Compare fragments from each of the different cutting enzymes. Record

 your results.

Assessment:

Students must keep a log of what they did each day and the purpose of the steps. They must record and analyze their results in comparing the DNA cuts using different restriction enzymes.

 

This lab was adapted from the access excellence web site

http://www.accessexcellence.org/AE/AEPC/WWC/1993/comparsion.html

 

Reference Standards:    Content Standard A & C