Genetics 2250
Laboratory 4: DNA extraction and amplification
2013
Part 1: DNA extraction
In this laboratory you will be using a rapid DNA extraction technique. There are several different
extraction techniques, the steps of which may differ, but the major chemicals and intent are the
same. The cells must be lysed to extract the DNA, and the cellular debris and inhibitors must be
removed. The Tris acts to buffer the solution, SDS is a detergent that lyses the cell membranes
and proteinase K removes proteins (e.g. histones). The chelex binds divalent cations (Ca2+ and
Mg2+) thereby disrupting normal cellular enzymes (e.g. DNase) from working.
Wipe your bench with ethanol at the beginning and end of the extraction. Wear gloves while
carrying out the procedure.
1. Label a 1.5 mL eppendorf (micro centrifuge) tube with the species name (cod), initials,
and date. It must be legible.
2. Cut a small piece of tissue from your piece of cod (~25-50mg, ~ size of a pencil eraser or
smaller). On a glass slide, cut the tissue into smaller pieces so that as much surface area
as possible is available. This will speed up the digestion process. Place the cut tissue in
the labeled 1.5 mL tube.
3. Locate the extraction buffer which contains chelex, on the side bench. There is one tube
of buffer that must be shared with the other groups. You will need to mix the buffer
frequently to mix up the chelex beads which have a tendency to settle at the bottom. To
make it easier to transfer the chelex, cut the bottom 3-5 mm off the P-1000 tips (this will
give them a wide bore). Add 300 l extraction buffer.
4. To the tube 3 µl of preteinase K (add directly into the buffer i.e do not drip it from
above). Close the tube immediately after adding the proteinase K. Mix by vortexing for
10 seconds in the vortex machine located on the desktop.
5. Incubate for 15 minutes in the 60C water bath.
6. Take the tube out of the 60C water bath. Vortex for 5 s to mix well. Centrifuge at 10,000
rpm for 1 min to pellet chelex and cell debris.
7. Label a new 1.5 mL eppendorf tube. This will be the tube that the DNA remains in.
Clearly label the top and side with the species name, your initials and the date. Locate the
1x low TE buffer on the side bench. Add 300 l of 1x low TE buffer.
8. Transfer 200 l of supernatant from the original tube to the newly labelled tube and try
not to transfer any of chelex that is at the bottom of the first tube. The chelex and
sediment at the bottom of the first tube has the lysed cells and debris that you want to
leave behind.
9. The liquid in the new tube now contains your extracted DNA. This can now be used for
downstream applications (e.g. PCR as in part 2). If you were not using the DNA it could
be stored for later use at -20C, however repeatedly freezing and thawing your samples
can damage the DNA.
Part 2: Polymerase Chain Reaction
In this section of the lab you will amplify the DNA that was extracted in part one. The PCR
technique is an invaluable technique which allows DNA to be quickly and easily amplified by
several orders of magnitude. By choosing the appropriate primers a specific sequence of interest
can be amplified, even if that sequence is present in low concentrations in your original solution.
1. In the fridge there are three 0.5 Eppendorf tubes labeled Gm1fr, Gm2fr, and Gm3fr. The
names of the tubes indicate the forward and reverse cod mtDNA primer in the tube. In
addition to the cod primer, the tubes contain buffer, dNTPs, and stabilized Taq.
2. Add 10 µl of the DNA that was extracted in Part 1 to each tube. Use a new pipette tip for
each tube.
3. Put your labeled samples into the PCR cycler. The machine has been preset for you. The
machine will start at 95 °C for 5 minutes, and then the temperature will cycle from 95 °C
to 50°C to 72°C and then back to 95 °C. This cycle will last ~ 2 minutes (including the
time needed for temperature shifts) and be repeated 15 times (this is for brevity due to
time constraints in the lab period; normally the cycle would be completed ~ 35 times).
Observe the machine for at least one temperature cycle.
4. When the lab instructor indicates that the programmed PCR cycle is finished retrieve
your tubes immediately and move on to part 3.
Part 3: Restriction Enzyme Digest:
In this section of the lab you will digest the DNA that was amplified by the Gm1fr primer with
ReII, a type II restriction endonuclease.
1. After the PCR process is completed, add 5 µl of the DNA that was amplified using the
Gm1fr primer to the tube in the fridge labeled REII, this tube contains a restriction
endonuclease
2. Put the tube labeled REII in the 37 °C water bath for 20 minutes (this waiting time is also
shortened due to time constraints, usually the reaction would be left for ~ 2 hours)
Part 4: Gel Electrophoresis of PCR product and RE digest
Gel electrophoresis will now be used to separate the fragments of DNA from the restriction
digest and the fragments that were amplified by the PCR process. An electric field is set up, and
since DNA is negatively charged the molecules move through the agarose matrix towards the
positive terminal. Small fragments move more quickly than larger fragments, thus the fragments
can be separated by molecular weight.
NOTE: The gels contain a very small amount of a fluorescent dye, ethidium bromide.
Ethidium bromide works by binding (intercalating) inside the DNA double helix, and for
this reason is both a mutagen and a carcinogen. Wear gloves at all times when handling
the agarose gel, and be careful not to spill any on yourself!
NOTE: Steps 1 to 4 have already been completed for you by lab staff to save time.
1. Place the gel tray in the electrophoresis tank with the gasketed ends forming a seal
against the walls of the tank. Be sure the tray is pressed all the way in and is level. Place
the comb in the end slot.
2. Wearing gloves, obtain 30 ml of molten gel (2% agarose in TBE buffer) from the hot
water bath. Slowly pour the agarose onto the center of the tray and let it flow to the edges.
Gently rock the tank back and forth to spread the gel out evenly. Leave the gel to cool
until set (~20 min).
3. When the gel is set, remove the gel tray from the running tank, turn it 90˚ so that the ends
are open and return it to the tank, making sure it is pressed all the way in and is level.
Also, ensure that the gel tray is in the tank with the wells (where the comb is located)
towards the negative (black) lead.
4. Fill the electrophoresis chamber with TBE buffer to ~ 1 cm below the fill line. Remove the
comb from the gel and place in the bowl provided; add enough TBE buffer to reach the fill
line (do not fill above the fill line).
5. Add 5 µl from each of the 3 tubes from the PCR (Gm1fr, Gm2fr, and Gm3fr = 15 µl) to
the tube labeled A which contains a loading dye. Mix the contents in the tube with the
pipette and add 15 µl to a gel well (be careful not to stab or tear the gel while adding the
sample)
6. Add 10 µl of the product from the restriction enzyme digest to tube B, which also contains
a loading dye. Mix the contents and add 15 µl to a gel well.
7. Sliding the cover on the tank, connect the negative lead (black) to the loaded end of the
box and connect the positive lead (red) to the other. [DNA is negatively charged & moves
toward the positive pole].
8. Electrophorese at ~100mA for ~30 minutes or until the blue dye has almost run off the
end of the gel. Make sure you turn off the power supply before removing the cover!
9. When the electrophoresis is finished, prepare to view your gels. Cover the filter of the UV
trans-illuminator with plastic wrap. Slide the gel onto the plastic wrap (avoid bubbles).
Switch on the trans-illuminator: the UV radiation causes the ethidium bromide to
fluoresce.
Caution: Prolonged exposure to the UV light can produce a sun burn in unprotected skin. Ensure
that the protective cover is on the trans-illuminator before turning it on and only keep the
machine on as long as necessary.
Questions:
1. Besides amplifying DNA in a PCR, what are 2 uses of extracted DNA? (0.5)
2. Explain the role of polymerase and primers used in the PCR reaction. (1.0)
3. At which end of the target sequence should the primers bind? Why? (0.5)
4. What does Taq stand for? Where is it found in nature? Why is Taq used instead of another
polymerase? (1.0)
5. Explain what occurs at each of the temperature steps in the PCR cycle. (1.5)
6. Approximately how many fold increase of genetic material would we get if we ran the PCR
for 15 cycles? (0.5)
7. You ran the gel electrophoresis without a control. Describe two potential controls or checks
that you could run on your gel. (1.0)
8. What is a Type-II restriction endonuclease? Describe an important application of restriction
endonucleases. (1.0)
9. Go to www.mun.ca/biology/hbennett and observe the pdf titled “restriction enzyme digest”.
Open the document using Foxit Reader and under view choose toolbars and then the
measurement tool bar. From this tool bar choose the distance function. Measure from the top of
the image to the middle of each band. The sizes (in base pairs) of all but one of the bands have
been provided for you. Graph the size vs distance travelled in Excel or Open Office Calc. Add a
logarithmic trendline to the graph, and extrapolate from this trendline to find the size of the
unknown fragment. Don’t forget to include an informative title and label the axes. (3.0)