Laboratory
Biol Biochemistry 100K
Jeremy Lee
Spring 2012
Lab 5 Protocol #1:
Agarose Gel Electrophoresis of Restriction Digests
and Undigested Plasmids
This protocol is similar to the protocol you used for your other gels; however, pay
attention to these specifics:
1. The % agarose in the gel;
2. The volume of gel and volume of EtBr added to the gel;
3. Since your digests are 20 ul, the volume you load in the gel, and the volume of
track dye you will add to your samples may also differ from previous gels.
PLEASE NOTE: THE TA WILL BE ADDING THE ETHIDIUM BROMIDE
TO YOUR GEL SOLUTIONS FROM NOW ON
ALL STEPS BELOW MUST BE PERFORMED WITH LAB COATS, GOGGLES,
AND GLOVES.
1. Pouring the gel: 50 ml of 0.9% agarose dissolved in 1X TAE buffer.
a. Weigh out the appropriate mass of agarose and add to 50 ml 1X TAE in a flask.
b. Loosely plug flask with paper towel and heat in microwave oven
approximately 1 minute, or until agarose is fully dissolved.
c. Remove flask from microwave and allow to cool in 55oC water bath for about 5 min
(you should then be able to comfortably hold it in your gloved hands for 10
seconds); do not allow it to begin to gel.
d. YOUR TA will carefully add 2.5 ul of 10mg/ml ethidium bromide (EtBr); swirl the
solution gently and slowly to mix (avoid getting bubbles in the solution.)
CAUTION: EtBr is a mutagen and, therefore, a potential carcinogen; handle
all materials containing EtBR carefully and as instructed by the instructor.
e. Put comb into gel tray: the tray should be oriented in the gel box so that its ends
are blocked against the sides of the box.
f. SLOWLY pour agarose/TAE/EtBr mixture into gel tray to avoid creating bubbles.
g. Allow gel to solidify and cool for 20-30 min or until fully solidified.
2. Prepare samples for loading into gel
a. For each digestion reaction and the undigested samples: Add track dye
directly into the 20 ul of your sample (you will be running the entire sample
in the gel); add enough track dye so that the track dye will be at a final
concentration of1X. Pipette up and down a couple times to mix.
b. For DNA ladder: pipette the same total volume (as samples plus dye above) of DNA
ladder into a microcentrifuge tube. (The DNA ladder already has track dye in it)
c. You should have, therefore, a total of 10 samples ready to load in your gel (6 digested
plasmid samples, 3 undigested plasmid samples, DNA ladder)
3. Setting up the gel
a. Reorient solidified gel in the gel box, so that comb/wells are on the appropriate
end of the box (to which electrode should wells be closest?)
b. Pour 1X TAE buffer into gel box until gel it is fully covered (about 0.5 1 cm above
gel.)
c. Slowly and carefully remove comb. Remove any air bubbles with a pipette tip.
4. Loading and starting the gel
a. SLOWLY pipette each DNA + track dye sample into a separate well in the gel.
i. Pipette tip should be put down into the well, but be sure NOT to poke the
pipette tip through the bottom of the well.
ii. DNA ladder should be placed in a separate well, either on the left or right of samples.
iii. Be sure to keep a record of which samples were loaded into which lanes.
b. Once all samples are loaded, close the gel box and attach all electrodes to the
gel box and to the power pack.
i. Use color coding to be sure all electrodes are attached to the right place
(the convention is that wires to the negative electrode are black, and wires
to the positive electrode are red.)
ii. MAKE SURE ALL WIRES ARE FULLY CONNECTED BEFORE
TURNING ON THE POWER PACK.
c. Turn on power pack. Set on 60V or as instructed.
i. You should immediately see bubbles rising from the wires in the gel box
ii. Within a couple minutes you should see the DNA moving into the gel from
the wells.
5. Removal of gel and documentation
a. When the fastest-running dye of the gel running dye has moved about 3/4 of
the way down the gel, turn off the power.
b. BE SURE POWER IS OFF BEFORE REMOVING ANY WIRES OR
OPENING THE GEL BOX.
c. Remove wires and open gel box.
d. Carefully remove gel as instructed.
e. Place gel on the UV transilluminator.
f. BE SURE YOU ARE WEARING LAB COAT AND GLOVES AND THAT
SHIELD OR CAMERA IS COVERING GEL BEFORE TURNING ON OR
LOOKING AT THE UV TRANSILLUMINATOR!!!
g. Turn on UV transilluminator. Ethidium bromide bound to DNA will fluoresce
indicating bands of DNA.
h. Photograph gel with camera the as instructed.
i. Dispose of gel in the appropriate waste receptacle as instructed.
j. Discard all solid waste potentially contaminated with EtBr as instructed.
Biochemistry Laboratory
Biol 100K
Jeremy Lee
Spring 2012
Lab 5 Protocol #2:
Polymerase Chain Reaction (PCR)
1. In your PCRs, you will be using two primers specific for the pET41-EGFP recombinant
plasmids. One of the primers, pAD1sense is specific for the EGFP insert; the other primer,
pAD1anti, is specific for a region of the pET-41 vector that flanks the EGFP insert.
2. Your DNA templates in PCR will be the three plasmids you isolated last week, plus a
positive control pET41/EGFP recombinant plasmid that we have isolated, giving you a
total of four PCRs, one template in each of four PCRs. You will also run a negative
control reaction, giving you a total of five reaction tubes to set up. Determine what you
think would be the proper negative control reaction for this experiment and think about
what your negative control would tell you; well discuss this in lab.
3. Here are the stock reagents used for your reactions:
Nuclease-free H20
10X PCR Buffer (already contains Mg2+)
10 mM dNTPs
10 uM of pAD1sense primer
10 uM of pAD1anti primer
Nuclease-free water
5 Units/ ul Taq polymerase
Plasmid DNA preps (assume DNA concentration = 50 ng/ul)
4. Each PCR should have a total volume of 20 ul. Below, on the left are the final concentrations, or,
where appropriate, the final amounts, of each reagent youll need to have in each reaction. Before
you come to class, calculate the appropriate volumes of each stock reagent and water youll add
together for each reaction:
Reagent: Final Conc/Amount
Nuclease-free H20 (to 20 ul final)
1X PCR Buffer
800 uM dNTPs
0.5 uM pAD1sense primer
0.5 uM pAD1anti primer
100 ng plasmid DNA
2.5 Units Taq polymerase
Volume of Stock Reagent to Add
_______________ ul
_______________ ul of 10 X PCR Buffer
_______________ ul of 10 mM dNTPs
_______________ ul of 10 uM pAD1sense primer
_______________ ul of 10 uM of pAD1anti primer
_______________ ul of 50 ng/ul plasmid DNA
_______________ ul of 5U/ul Taq polymerase
5. Once your calculations have been checked by your TA, you will make your own master
mix of all the reagents that are common to all your reactions, i.e. everything except DNA:
i.e. buffer, dNTPs, primers, water, and, finally, Taq polymerase (which your TA will add to
your master mix after everything else has been added by you.) Here are some tips for
making your master mix:
A. Its generally a good idea to make a bit more master mix than youll actually need. Since
you will be setting up five reaction tubes, make enough master mix for six reactions.
B. To make a master mix for six reactions. multiply by six the volume of each reagent you
need per reaction (except DNA!) as calculated in step #4. Then add these total volumes
of reagents to a single tube (Taq polymerase will be added in the next step by your TA.)
Mix well by pipetting up and down. Then place your master mix on ice.
C. Once youve made your master mix, your TA will then add the appropriate amount of
Taq polymerase to your master mix. Its important to keep your master mix on ice
as much as possible at this point.
D. IMPORTANT: Mix your master mix well by slowly and carefully pipetting up and
down. Then aliquot (distribute) the appropriate volume of master mix for each reaction
to each of five tubes; KEEP ON ICE! (You should have some master mix left over.)
E. Add the appropriate DNA template to each of your five tubes (what should you do with
your negative control?)
F. Mix contents of each tube thoroughly by pipetting up and down slowly and carefully with
your micropipettor.
6. Place labeled tubes in the thermocycler. Once everyones tubes are in the thermocycler, your TA
will start it up, and it will be set to incubate your samples according to the following PCR
parameters:
95oC for 10 min
95oC for 1 min
56oC for 1 min
72oC for 1.5 min
(30 cycles)
72oC for 5 min (1 cycle)
4oC (hold) (to suspend reaction and prevent degradation of DNA)
7. We will remove your samples from the thermocycler and store them for you at 4oC. You will
analyze them by gel electrophoresis during next weeks lab. Next week in lab, youll also use
online DNA analysis tools to determine what size PCR products you expect to see in your gel.