Summer
2001
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