Laboratory Class 5: Nucleic acids 2025

PROCEDURES

In experiment 1 you will extract DNA from a homogenized calf liver sample. For experiment 2, a
solution of commercially purified calf DNA will be provided by the lab technician so that you can

analyze the effect of salt at low and high concentrations on DNA structure. Using the same
grity of the in-lab prepared
DNA with that of the commercially derived source of DNA. In experiments 3 and 4 you will be
introduced to the techniques of restriction digestion and DNA electrophoresis on agarose.

Before performing these experiments, you should watch the following videos:
DNA melting (5:19) or (https://www.youtube.com/watch?v=YV4nonUsb2Q)
Gel electrophoresis Part 1 (13:05) or (http://www.youtube.com/watch?v=3ukaT_Ih9d8)
Gel electrophoresis Part 2 (8:13) or (http://www.youtube.com/watch?v=_QxxB65Gi78)

You should also perform the following simulations:

Restriction Enzyme Digest
(https://www.labxchange.org/library/items/lb:LabXchange:1fb8b9d5:lx_simulation:1)
Agarose gel electrophoresis
(https://www.labxchange.org/library/items/lb:LabXchange:9548bee3:lx_simulation:1)
Verifying a recombinant plasmid by gel electrophoresis
(https://www.labxchange.org/library/items/lb:LabXchange:b740f160:lx_simulation:1)

EXPERIMENT #1: Isolation and characterization of calf liver DNA

a) DNA extraction

1. You have been provided with 600 mg of calf liver tissue in a 50mL Falcon tube. The calf
liver has already been homogenized (with a polytron) in 4 mL of cold Standard saline-
EDTA (0.15 M NaCl in 0.1 M ethylene-diamine tetra-acetate EDTA, pH 8.0 with
) in a 50 mL Falcon tube.

2. gently by inversion. (SDS is a

detergent and will foam extensively; you could also easily shear the DNA).

CAUTION! The SDS solution may irritate the skin and eyes. Use gloves and
safety glasses.

(http://ccinfoweb2.ccohs.ca/msds/Action.lasso?-database=msds&-layout=Display&-
response=detail.html&-op=eq&MSDS+RECORD+NUMBER=5502925&-search )

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3. Incubate the mixture in a 60°C water bath for 10 minutes and then cool to room
temperature.

4. Add 0.725 mL of 6.0 M NaClO4 and mix gently.

CAUTION! The perchlorate solution may irritate the skin and eyes. Use
gloves and safety glasses.
(http://www.sciencelab.com/msds.php?msdsId=9925018 )

5. Add 5.0 mL of chloroform:isoamyl alcohol (24:1, v/v) in the fume hood.

CAUTION! Chloroform and isoamyl alcohol are toxic and irritate skin, eyes
and respiratory tract. Keep away from sparks and flame. Use gloves and
safety glasses. Avoid inhalation. Work in the fume hood.
(http://www.sciencelab.com/msds.php?msdsId=9927133 )
(http://www.sciencelab.com/msds.php?msdsId=9927550 )

6. Mix gently on a waver for 5 minutes.

7. Centrifuge at 12,000 xg for 5 minutes. (Tubes should be balanced!).

8. Carefully remove 70% of the upper aqueous phase by aspirating the liquid off with a
plastic pipette and transfer it to a 15 mL Falcon tube. Avoid contaminating your sample
with the denatured protein that collects at the interface between the aqueous and
organic phases. Dispose of the chloroform phase in the appropriate waste container.

9. Gently layer 10 mL of 95% ethanol over the aqueous phase in the tube. Seal the tube
with its cap and mix gently and continuously until the ethanol becomes fully mixed with
the aqueous phase (Observe the precipitation of the DNA).

10. Centrifuge in the 15 mL tube directly at 3000g for 2 min (swinging bucket tabletop
centrifuge).

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11. Carefully decant the supernatant in the sink then add 2 mL of 70% EtOH to the pellet in
the 15 mL tube. Centrifuge again at 3000g for 2 min. Decant supernatant again carefully.
Remove as much EtOH as possible using a P100 pipette, then let the pellet air dry for 10
mins.

12. Add 2 mL of 15 mM citrate buffer to the pellet. Place the tube on the waver for 5 mins.

13. Transfer 1 mL to a 1.5 mL microtube and centrifuge it for 1 min at 13,000 rpm.

b) DNA characterization

You will determine your DNA yield as well as the degree of purity and integrity of a DNA sample
prepared as above.

14. Pipette 50 950

of 15 mM citrate buffer. Mix by inversion. Read and record the absorbance at 234, 260
and 280 nm of your sample with the UV-spectrophotometer. Do NOT zero the
spectrophotometer; the technicians will have already done it for you!

15. Prepare a dilution of your DNA sample with a final volume of 1mL in 15 mM citrate
buffer, pH 7.0 in order to have an A260 between 0.2 and 1.0. Calculate and record your
dilution factor. This solution will be used for the DNA melting curve experiment (step
18). Only prepare a second dilution if your A260 reading falls outside the range of 0.2-
1.0. If the dilution from step 14 falls within the range, your sample is ready for the
melting curve experiment.

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EXPERIMENT #2: DNA melting curves

For a melting experiment, you will follow the change in absorbance at 260 nm of the DNA in
function of the temperature. You will use the Cary100 Bio system that can analyse 12 DNA
samples simultaneously.

Melting experiments will be performed on the following DNA samples:

a) Calf DNA from a commercial source,

b) Calf DNA from a commercial source, in the presence of salt at low
concentration,
c) Calf DNA from a commercial source, in the presence of salt at high
concentration,
d) Calf DNA from step 15.

16. Prepare 1 mL of a 1:20 dilution from the stock commercial DNA solution (in the clear
labelled tube) commercial DNA solution and
orresponding buffer). Make sure to prepare your dilution in a
QUARTZ cuvette!

Note: All buffers have been de-aerated by sonication. This will prevent bubble
formation during the heating process.

solution a: commercial calf DNA in 15 mM citrate buffer, pH 7

(Teams 1, 2, 3, 8, 9, 13, 14, 15, 17, 19, 20 and 21)

solution b: commercial calf DNA in 15 mM citrate, pH 7, 0.03M NaClO4

(Teams 4, 5, 10, 11, 22 and 23)

solution c: commercial calf DNA in 15 mM citrate, pH 7, 4.75M NaClO4

(Teams 6, 7, 12, 16, 18 and 24)

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17. Once all samples are loaded into the Cary 100Bio spectrophotomer (as directed by your
TA), the technician will start the melting experiment.

18. Once experiment 1 is completed, each student will set up a second melting experiment
with your own extracted DNA sample.

19. Once the melting is finished, data corresponding to your 4 melting experiments
(absorbances and 1st derivative) will be exported in your dataset.

EXPERIMENT #3: Restriction Endonuclease Mapping of a Plasmid

In this experiment, an unknown plasmid (100 ng/µL) will be digested with a combination of two
different restriction enzymes: EcoRI (10 U/µL) and HindIII (10 U/µL). You will be supplied with
H2O, a solution of 10X digestion buffer (Buffer O) and an unknown plasmid (100 ng/µL). Each
student will be digesting a different plasmid.

20. Prepare the following reaction mixtures in 4 labelled 0.5 mL microcentrifuge tubes by
adding the following reagents in the order they are listed. Ask your TA for the EcoRI and
HindIII restriction enzymes, adding them to the mixture last; rinse the pipette tip with the
solution at least 5 times. Always keep your tubes on ice until loading it into the 37oC water
bath.

Table 1. Plasmid digestion mixture

EcoRI +
Reagents EcoRI HindIII Control
HindIII
H2O (brown tube) 11 µL 11 µL 10 µL 12 µL
10X Buffer (blue tube) 2 µL 2 µL 2 µL 2 µL
Unknown plasmid (100 ng/µL)
6 µL 6 µL 6 µL 6 µL
(Orange tube)
EcoRI (10 U/µL) (see TA) 1 µL ---- 1 µL ----
HindIII (10 U/µL) (see TA) ---- 1 µL 1 µL ----
Total volume 20 µL 20 µL 20 µL 20 µL

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21. Mix the solutions by tapping the bottom of the tubes with your finger. To bring all the
reagents to the bottom of the tube, centrifuge for 15 seconds. (Make sure your tubes are
balanced!)

22. Incubate your tubes in the 370C water bath for 45 minutes.

23. Once the digestion is completed, add 2.2 µL of 10X DNA-electrophoresis loading buffer to
each sample. Mix thoroughly.
Proceed now with electrophoresis on the provided gel (step 29).

EXPERIMENT #4: Agarose gel electrophoresis

N.B. To avoid waiting time, a gel for every student is provided. Your TA will demonstrate
how to prepare a gel (steps 24-28).

24. In a 250 mL bottle, prepare 100 ml of a 1% (w/v) agarose solution in TAE (40 mM tris-
acetate, 1.0 mM EDTA) buffer. Check calculation with your TA before proceeding.

25. Heat the agarose suspension in a microwave oven. To prevent pressure from building up
inside the bottle, loosen the cap before heating. Use high setting 3 x 1 min, mixing after
each minute. Make sure agarose is completely melted.

26. Once dissolved, allow to cool to 50-55°C (agarose solidifies at about 40°C); any hotter
than 55°C will melt the gel casting mold.

27. When the gel solution is at 50-55°C, add 10 µL of SyBr Safe DNA Stain.

28. Place the gel running tray in the gel casting mold and pour the cooled (50-55°C) agarose
solution into the casting mold. Pour slowly to avoid making bubbles. Place a 20 well

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comb at 1 cm from one end of the casting tray. Dislodge any bubbles with a pipet tip.
Allow the gel to solidify for at least 20 min.

29. Remove the comb by slowly lifting one end, making sure not to suck the agarose out of
the bottom of the well.

30. Lift the gel tray from the casting mold and place in the electrophoresis chamber. Pour in
the TAE buffer until the gel is covered by 4-8 mm of buffer.

31. Load 10 µL of provided MassRuler Express Forward DNA ladder marker (M) and 15 µL of
the samples from step 23. Place the end of the tip below the surface of the running
buffer immediately above the desired well. Expel the sample slowly (taking care not to
allow any air bubbles to form under the tip), the high concentration of glycerol in the
loading buffer will cause the entire sample to sink to the bottom of the well.

Gel loading template

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32. Place the lid on the electrophoresis unit and connect the electrodes such that migration
proceeds towards the RED (positive, anode) electrode. Connect the leads to the power
supply and apply 100 V for 45 min.

33. Turn the power supply off, disconnect both ends of the electrodes and remove the gel
tray. (WEAR GLOVES)

34. Place the gel and running tray on the UV Gel Doc System and take a picture. (Ask your
TA for help). The file corresponding to gel as well as the loading order will be provided
in your dataset.

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RESULTS AND DISCUSSION

R1. From the UV absorption data comment on the purity of your DNA preparation.

DNA melting curves

R2. Show the 4 graphics with the melting curves (absorbance and 1 st derivative) for DNA in
different conditions (samples a to d). From the 1st derivative plots, estimate the
corresponding Tm values. From the absorbance plots, estimate the values of h(%) and T.
Show your calculations. Summarize your results in the following table.

Table 2. DNA melting results

DNA sample Tm (°C) T (°C) h (%) h/ T (°C-1)

a) Commercial
b) Commercial + 0.03M NaClO4
c) Commercial + 4.75M NaClO4
d) Extracted DNA (step 15)

R3. From your results R2, discuss the effect of salt at low and high concentration on the DNA
structure. Tm and hyperchromicity (h) should be discussed.

R4. From your results R2, comment on the purity and integrity of the extracted DNA. Tm and
hyperchromicity (h) should be discussed.

Restriction Digestion and Gel Electrophoresis

R5. Show a figure with the picture of your gel (step 33) and the corresponding labels. Estimate
the migration distance of all bands. Plot logMW (bp) of the MassRuler DNA ladder (Figure
3 and Appendix C5) vs. the migration distance (show the figure). Estimate the MW (bp)
of all DNA fragments in your three digested samples using the equation generated from
the graph.

R6. From the number and size of the fragments observed in your three digested samples,
create a map of the plasmid showing the relative location of the restriction sites for EcoRI
and HindIII.

R7. Looking at your gel electrophoresis identify the conformation(s) that your undigested
plasmid has adopted. Were your digestions (EcoRI and HindIII) complete? Why or why
not?

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REFERENCES

1. Boyer, R. (2000). Modern Experimental Biochemistry. 3rd Ed. Benjamin Cummings, p

399.

2. Winfrey, M.R., Rott, M.A. and Wortman, A.T. (1997). Unraveling DNA. Molecular Biology
for the Laboratory. Prentice Hall (N.J.), pp 42 and 56.

3. Voet, D. and Voet, J. (2004). Biochemistry, 3 rd Ed. John Wiley & Sons, 5.3C, p 90.

4. Bergeron-Wiper, N and Rodriguez M. (2004). Electrophoresis.Tutorial notes IV.

5. Voet, D. and Voet, J. (2004). Biochemistry, 3rd Ed. John Wiley & Sons, 6.4, p 144.

6. Voet, D. and Voet, J. (2004). Biochemistry, 3 rd Ed. John

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