Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
Procedure 1: DNA Isolation from onion cells
First make sure that you have all the necessary tools and materials ready for use on your
bench by check marking the Equipment & Materials list below
Necessary Equipment:
Check Mark ( √ )
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Pistil & Mortar
Table balance
Spectrophotometer
Bunsen burner & lighter
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Adjustable-volume pipette
Pipette pump
Glass beaker (50ml, 250ml)
Cheese cloth
Graduated glass cylinder (10ml)
Glass pipettes (5ml, 10ml)
Glass rod
Petri dish
Glass marbles
Glass funnel
1.5ml reaction tube (PP, Eppendorf type)
10ml test tubes (tempered glass)
reaction tube rack
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Required Materials & Reagents:
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Double-distilled water (= ddH2O)
2 yellow onion bulbs
Meat tenderizer (papain powder)
Sodium chloride (NaCl)
Saline citrate buffer
100% Ethanol p.a. (ice-cold)
Diphenylamine (DPA) stock solution (5mg/ml)
Bovine serum albumin (BSA) stock solution (30mg/ml)
Salmon sperm DNA
10% SDS solution
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Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
Procedure:
a. Cut two pieces of yellow onion about one-half inch cubed and place them in
mortar
b. Prepare 10 ml of a cell lysis solution by adding 1 ml of a detergent solution
(10% SDS) and 1 g of NaCl (use the balance) to a 10ml graduated cylinder and
fill it to the 10ml mark with distilled water
the detergent SDS disrupts the phospholipid membranes of the onion cells
therefore releasing the cell contents including DNA and proteins
c. Macerate the onion pieces slowly with a pestle by avoiding formation of
bubbles
- maceration mechanically breaks the cell walls, allowing the membranedissolving detergent to disrupt the cell membranes and to release the cell
contents
d. Filter the mixture from the mortar through a clean cheese cloth into a clean
50ml glass beaker
- large pieces of cellular material will be filtered out and trapped in the cheese
cloth while most of the DNA will enter the beaker
e. Using a 5ml glass pipette, transfer 4ml of a 5% (w/v) papain solution to the
beaker and gently swirl periodically for 5 min to mix the contents
- Papain is a protease isolated from papaya fruit which digests cellular proteins
including the proteins which are attached with genomic DNA, called histones
- papain is also used in meat tenderizers to break down meat proteins making
meat more tender
- freshly prepare the 5% papain solution by weighing in powdered papain and
dissolve it in 4ml of distilled water
- show your calculations in question 4 (Q. 4) of the Section Questions part at the
end of this lab manual
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What does (w/v) stand for? ___________________________
f. Pour the combined solution of papain and onion cell lysate into a clean Petri
dish and place the dish over a dark surface
- the Petri dish is a very common lab glass ware routinely used by microbiologists
to cultivate bacteria and other microbes
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the dish is named after its inventor, Julius Petri (1852 – 1922), a German bacteriologist
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the dark surface simply makes it easier to see the DNA as it becomes visible
after its precipitation with ethanol
g. Now, using a clean 10ml glass pipette, slowly add 10 ml of ice-cold 100%
ethanol p.a. (ethyl alcohol) along the edge of the Petri dish. Look for whitecolored clumps to form in the dish which is your precipitated DNA!
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ethanol, as a mild organic solvent, expels the water which surrounds the DNA
like a cloud (= Hydration layer) and triggers DNA precipitation
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Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
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as a result of this “dehydration”, the previously single DNA strands start to clump
together (precipitate) forming a network of millions of long, white DNA strands
the story is actually a bit more complicated; I refer to Appendix B if you are
more curious about why and how alcohol precipitates DNA
h. Spool DNA on to the end of a clean glass rod which you swirl in the Petri dish
- the negative charges (-) of DNA’s phosphate groups in the sugar phosphate
backbone (see DNA model) become attracted to the positive net charges of the
silica groups of the glass rod
- as you stir the glass rod within the cell lysate solution more and more DNA
molecules will begin sticking to the end of the rod
i. Transfer some of the spooled DNA on your rod into a clean 1.5 ml reaction
tube and save it in a freezer for later use (see DNA digestion &
Electrophoresis lab)
- you will need this material to digest it with restriction nucleases and to separate
the thus digested DNA with the help of the agarose gel electrophoresis method
j. Transfer some of the spooled DNA on your glass rod into two clean 10 ml
glass test tubes and place it into a rack on your bench
- you will need the material in one tube to proof that it is indeed DNA you just
isolated following the steps of the Qualitative Method as described in Section 2
of this lab manual
- you will need the material saved in the second tube to determine the amount of
DNA collected following the procedure as described in Section 3 below
k. Touch the remaining material on the rod with your fingers to get your
(probably first-time) “hands-on” experience with the hereditary molecule of
life on planet Earth … with DNA!! You did it! Congratulations!
DNA
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Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
Lab Assignment 1: Colorimetric identification of DNA using diphenylamine (DPA)
1. Test the material you have collected in your test tube with DPA by following the
steps 1  6 as shown in Graphic 1 on the next page and record the color
change in the “Results” table below
- add 3ml of saline citrate buffer to your spooled and collected onion cell
material (= sample reaction)
- mix thoroughly by shaking
2. Set up in parallel four, so-called control reactions in other test tubes to
minimize the danger of false conclusion. Follow the experimental outline as
depicted in Graphic 1 below. Observe and record eventual color changes in the
Result Table 1 below
- the positive control reaction contains 9 – 10 mg salmon sperm
DNA dissolved in 3ml of saline citrate buffer
- one negative control reaction will contain no DNA and only 3ml
of saline citrate buffer
- a second negative control reaction will contain 9 – 10 mg of the
protein BSA dissolved in 3ml of saline citrate buffer
Result Table 1:
Monitor the color changes in your test tubes and record your observed results in the table
below.
Write the content of your test tube (= Step 1) into the bracket of the first (left) column
Test Tube No.
Color
(after step 5)
Interpretation &
Conclusions
#1
(
)
#2
(
)
#3
(
)
#4
(
)
#5
(
)
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Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
Graphic 1: Experimental Test Tube Set-Up
Lid test tubes
with glass
4
?
?
Tube #
1
2
3
4
5
1
Content
Onion
DNA
Onion
DNA
Sperm
DNA
BSA
Blank
2
Citrate Buffer
3
6
3
3
3
DPA
3
0
3
3
3
3
[ ml ]
Graphic©E.Schmid/2003
o
5 Heat for 10min at 95 C
6
Monitor & Record the color reaction
Lab Notes:
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Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
Lab Assignment 2: Quantitation of DNA using diphenylamine (DPA)
The standard method of molecular biologists to measure the amount (quantity) of DNA in a
given solution is to measure the absorbance of the DNA molecule of ultraviolet (UV)
light at a wavelength of 280nm (= A280 value).
 for those of you more interested rehearse the physical nature of light
and its different wavelength of the typical light spectrum!
This method however requires an expensive, high-precision machine, called a UV
spectrophotometer, which we unfortunately do not have available in this lab course.
But fortunately we are in possession of a spectrophotometer which is able to measure
the absorption of molecules in the visible (VIS) light spectrum. We will use this sensitive
precision equipment to measure the characteristic light absorption of the DNA indicator
dye DPA after binding to DNA. Remember, DPA after reaction with DNA forms a deep
blue colored compound which strongly absorbs (interacts with) visible light with a
wavelength of 660nm.
 rehearse the components and function of a spectrophotometer
 how does it work?
 what is the difference between light absorption and transmission?
This part of the lab requires the establishment of a so-called standard curve, which
requires exact knowledge of the quantity of your standard DNA (in our case: Salmon
sperm DNA
The standard DNA has to be exactly weigh in, dissolved in an exactly defined volume and
requires good pipetting skills since you have to make serial dilutions starting with the socalled Standard DNA Stock solution (see Graphic 2 below)
Procedure:
a. Place 7 clean glass test tubes in a rack and label them 2 through 8 using a
permanent marker pen (see pipetting scheme in Graphic 2 below)
b. Weigh in 30mg of Salmon sperm DNA into a clean 15ml plastic centrifuge
tube (with screw cap), pipette 10ml of saline citrate buffer to this tube and
dissolve the DNA by gently shaking or swirling the closed tube
 What is your final concentration of Standard DNA in this tube?
DNA Standard Concentration cSt = ______ mg/ml
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Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
Graphic 2: Quantitative DNA Assay using the DNA Indicator Dye DPA
Cover test tubes
4 with ParafilmTM
etc.
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Test Tube #
DNA
1
2
3
4
5
6
7
8
Salmon Sperm*
Onion
Blank
1
DNA-Stock
Sample
3
1.5
0.6
0.3
0.15 0.06
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2
Citrate Buffer
3
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1.5
2.4
2.7
2.85 2.94
3
3
3
3
3
DPA
3
3
solution
Dilution
factor
undil.
undil.
DNA- [μg/ml]
Conc.
?
3
3
[ml]
3
Graphic©E.Schmid/2003
5
Incubate overnight at 25 - 30oC
6
Spectrophotometer
Read the absorption of the
DNA standard & Sample DNA at 660nm
 Prepare a standard curve
c. Label the test tube with the onion DNA sample you collected and set aside in
Section 1 above as number “1” , pipette 3ml of saline citrate buffer to this
DNA and dissolve it by gently swirling the test tube; place in rack next to the
other tubes
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Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
d. Pipette 3ml of the DNA standard solution into tube #2 and do the serial
dilution of the DNA standard following the pipetting scheme as shown in
Graphic 4 below
 Calculate the dilution factors and the final DNA concentrations
of the DNA standards in test tubes #3  #7 and write the
numbers into the corresponding boxes of Graphic 4
e. Pipette only 3ml saline citrate buffer into test tube #10
 this test tube functions as _________________ during this
experimental set-up
f. Pipette 3ml of DPA solution to all 10 test tubes, mix the contents by gently
swirling the tubes
g. Cover all tubes with a piece of ParafilmTM to avoid evaporation and incubate
them (placed in a rack) overnight at about 25 - 30oC
- this procedure is safer, about three times more sensitive and less prone to
artifacts (e.g. false positives) than the previously applied “boiling method” (see
Section 2)
h. Turn on the spectrophotometer, adjust the wavelength to 660nm and measure
the absorbance of the contents of each test tube (1  10) using 3ml plastic
cuvets
 your instructor will show you how to operate the spectrophotometer
and how to proceed with the absorbance readings
 Rinse the cuvets with distilled water in between each new reading!
Quick questions
- Which tube will you begin with? Test tube # _____
- Why? ___________________________________________________
i. Write the recorded absorbance readings at 660nm wavelength (= A660) for
each of the 8 test tubes into the Result Table 2 below
Result Table 2: Absorbance readings of the DNA quantitation assay
Tube #
1
2
3
4
5
6
7
8
Absorbance
[A660nm]
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Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
Results discussion
(Student Name)

Use a PC and a suitable data management program, e.g. Excel or SigmaPlot, to
prepare a standard curve using the retrieved A660 values of your DNA standard at
the different dilutions
 print your resulting graph on paper and put your name on it!
 this document is an important part of your weekly lab report!
 if your are not a computer wiz, you can of course simply use a
piece of graduated paper and draw your standard curve manually
using a pen or pencil
Questions:
- Which of your data in go on the abscissa (= x-axis)? (circle)
A) dependent parameter
B) independent parameter
i. DNA Concentration
i. DNA Concentration
ii. Absorption (A660)
ii. Absorption (A660)
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Which one on your ordinate (= y-axis)?
____________________________
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Do you observe a linear correlation of your data?
____________________________________
Use the above established standard curve and its corresponding linear equation
(Excel and other programs will tell you that after you typed in the data and
performed a “linear regression” operation)
(1) A660(cSt) = a cSt + b
to determine the exact concentration of your onion DNA in your
test tube #1
 it is important at this step that you determine and know the size
of your variables “a” and “b” of your linear equation!
a = ________
b = ________
j. Knowing the A660 value for your onion DNA in tube #1 and using equation (1)
above, calculate the concentration of your onion DNA (= co-DNA) in tube #1
now
k. The calculated onion DNA concentration in test tube #1 is:
Co-DNA = A660(tube#1) – b = _______ mg/ml
A
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