BC2004
Lab Exercise 11
Spring Semester 2005
Gel Electrophoresis of DNA
weeks of 4/18-4/22 and 4/25-4/29/05
Gel electrophoresis is the most widely used method in molecular biology for separating
macromolecules from one another on the basis of size. It is especially useful for analyzing
mixtures of proteins or of nucleic acids with respect to the presence and relative abundance of
molecules of different sizes, and for estimating the size of purified macromolecules; it can also
be used as a step in purification of a specific protein or a specific length-class of DNA.
Electrophoresis gels for nucleic acids are most commonly prepared with agarose; gels for
proteins are most commonly prepared with polyacrylamide. In this laboratory, agarose gel
electrophoresis will be used to separate DNA molecules that differ in number of nucleotides and,
therefore, in length (size). With a few changes in details (gel preparation, buffers used, stains
applied, etc.), the same general approach would used be separate proteins on the basis of
molecular weight on a polyacrylamide gel.
The fundamental principle of gel electrophoresis is that charged macromolecules will migrate
through a gel when an electrical field is applied. Remember that DNA (in a neutral or basic
environment) is negatively charged from the O- on the phosphate groups of the sugar-phosphate
backbone. Because DNA is negatively charged, it will move toward a positive electrode and
away from a negative electrode.
The distance migrated by a DNA molecule during the time it is subjected to the electrical field is
determined by three factors: 1) the size (length) of the molecule; 2) the electrical field
(dimensions and voltage differential); and 3) the density of the gel matrix. When a sample
containing DNA molecules of different sizes is applied as a mixture to the same position in the
gel, all of the molecules are subjected to the same voltage differential, and all of the molecules
are challenged to work their way through the same gel matrix. This leaves molecular size as the
only factor that will determine the final relative positions of the DNA molecules present in the
original mixture.
In environments of pH greater than 7, DNA molecules carry an abundance of negative charges
that are uniformly distributed along the length of each molecule. When subjected to an electrical
field under neutral or basic conditions (with a pH > 7), DNA molecules migrate toward the
positive pole (the anode) of the electrical field. In a gel matrix, the movement of DNA molecules
in response to the electrical field is impeded by the gel, which acts like a sieve that slows the rate
of their migration.
The larger the molecule, the more difficult it is for the molecule to work its way through the gel,
so that smaller molecules move through the gel faster than larger molecules. As smaller
molecules get ahead of larger molecules, they also get closer to the anode, and their migration
accelerates in proportion to their proximity to the anode: the closer the molecule gets to the
BC2004, Spring Semester 2005, Lab Exercise 11-1
anode, the faster it migrates. Consequently, as smaller molecules of DNA get ahead of the larger
molecules, the relative rate of migration of the smaller molecules is determined by two factors:
(a) their relative ease of working their way through the gel matrix, and (b) their increasing
proximity to the positive pole of the electrical field. This compounding of influences on the rate
of migration results in an exponential relationship between molecular size and distance migrated
(see standard curve construction, below).
Gel electrophoresis results in the separation of DNA molecules of a mixture according to their
molecular length (size), with the smaller molecules moving ahead of the larger molecules, which
trail behind the smaller ones in order of increasing size. Wherever there are many DNA
molecules of very nearly the same size, those molecules accumulate as a migrating “band” that is
separated from smaller molecules ahead of them in the lane of their migration, and larger bands
behind them.
The positions of the bands can be visualized by staining the gel with a dye that binds to DNA
molecules, and the size of the molecules in each band can be estimated by comparison of the
band’s position (distance migrated) with the positions of DNA molecules of known size. In this
technique, a standard curve serves as the means of estimating the size of the molecules, in
parallel to the use of standard curves for estimating the concentration of smaller molecules, as
you learned in Exercises 1 and 2 of this laboratory course.
There is, however, one notable difference between the standard curves of Exercises 1 and 2 and
the standard curve that you will construct in this exercise: the relationship between absorbance
and solute concentration (in the useful range of the standard curves) was linear, whereas the
relationship between molecular size and distance migrated in an electrophoretic gel is
exponential, as explained above. For this reason, the standard curve that you construct in this
exercise will be plotted using semilogarithmic coordinates so that the curve generated will relate
the logarithm of molecular size to distance migrated plotted on a linear scale.
In this exercise, you will analyze DNA molecules in five preparations – two PCR reaction
mixtures from Exercise 9 and three restriction endonuclease digests from Exercise 10. You will
first mix your DNA samples with a dye called SYBR-Green, which binds to DNA and fluoresces
when exposed to the proper wavelengths. You will then load each of your five DNA samples
and a solution of DNA molecules of known sizes (your “Markers”) into wells in a gel prepared
with 0.7 % agarose in 0.5 X TBE buffer, mounted in an electrophoresis apparatus and submerged
in 0.5 X TBE buffer at pH 8. When all preparations (six for your group and six for another group
of students) have been loaded into your gel, the electrophoresis apparatus will be closed,
electrical leads will be attached, and sufficient current will be applied to maintain a voltage
differential of approximately 120 V across the gel.
Electrophoresis will continue until the indicator dye approaches the positive end of the gel. This
will probably require approximately 45 minutes. After the gel has been run, you will disconnect
the electrical leads and expose the gel to the appropriate wavelengths of visible light. These
wavelengths will cause the SYBR-Green (bound to the DNA) to fluoresce. You will mark the
location of the fluorescent bands of DNA and estimate their length in base pairs.
BC2004, Spring Semester 2005, Lab Exercise 11-2
Credits: The restriction digestion performed in Exercise 10 and analyzed here is based on a
procedure developed by the DNA Learning Center of Cold Spring Harbor Laboratory that
accompanies Carolina Biological Supply Company’s Restriction Mapping of Lambda DNA Kit
(catalog no. 21-1173).
Procedures
Do Parts B-D first. Your instructor will then demonstrate gel preparation. Following the
demonstration, prepare one gel (Part A) for each group of 4 students (2 pairs). At some point
during the lab period, your instructor will demonstrate how to transfer a gel to the visualization
light box (you’ll need to be VERY careful because your gel is VERY fragile).
A. Preparation of an agarose gel.
Because your time in the lab is short, the gel you and your partners use will be ready for you at
the beginning of your lab period. Nevertheless, in order for you to learn the steps in gel
preparation, you and your partners will prepare one gel that will be used by the next lab section.
1.
Close the ends of a gel tray with tape. The attachment must be watertight, so press the
tape firmly against the ends of the tray. Insert the comb that forms the wells at one end of
the tray.
2.
Weigh 0.7 g agarose onto a piece of weighing paper (it works best to fold the paper in
half and then open it up again so that you can easily pour the powder off the paper
without spilling it; don’t forget to tare the balance after putting on the paper and before
adding the agarose). Transfer your weighed agarose into a 250-ml Erlenmeyer flask.
Measure 100 ml 0.5X TBE buffer in a graduated cylinder. Pour the buffer into the flask
and mix by swirling gently.
3.
Close the flask with a wadded up KimWipe or two (you’ll need to leave some room for
air to come up so the flask doesn’t explode in the microwave) and heat it for 1 minute in
the microwave oven.
4.
Using a hot-glove to protect your hand, remove the flask and gently swirl the contents.
Examine the contents to be certain that the agarose is completely melted; if it is not (and
the solution looks even a little bit grainy), return the flask to the oven for additional
heating. When the agarose has been completely melted, remove the flask to a water bath
at 65o C to cool for a few minutes.
5.
When the molten agarose has cooled to approximately 65o C, swirl it gently to be certain
that the agarose is evenly distributed, and then gently pour the contents of the flask into
the prepared tray. Rupture any air bubbles by poking them with the top of a clean pipet
tip while the agarose is still molten.
6.
Allow the gel to stand until it appears to be firm and is slightly cloudy, and then turn in
the gel for use by the next lab section.
BC2004, Spring Semester 2005, Lab Exercise 11-3
B. Preparation of the DNA solutions.
Your 2-PCR mixtures and 3-restriction digests have been stored in the freezer since your lab
exercise 9 and 10 sessions. Retrieve them from your instructor and examine them to be certain
that they have thawed completely.
1. Restriction digests. Using a fresh pipet tip for each addition, place 10 μl 3X SYBR-Green
Loading Buffer into each digest tube. Mix the contents by gently aspirating the mixture into the
pipet tip and then expelling the mixture back into the tube – once only. Avoid bubbles!
Lambda DNA is predominantly two-stranded, but when it has been cut, it will have one-stranded
sticky ends. As a consequence, some of the molecules in your digests will reassociate with each
other, making fragments unsuitable for accurate determination of restriction fragment length. In
the absence of DNA ligase, as in our mixtures, these reassociated molecules are held together
only by hydrogen bonds, which can be disrupted by heating the digests at 65oC for 10 min.
Therefore, heat your dye-digest mixtures in the 65oC incubator for 10 minutes immediately
before loading them into your gel.
tube
Digest A
Digest E
Digest A/E
amount of SYBR-Green
Loading Buffer
10 uL
10 uL
10 uL
amount and identity of restriction
digest volume
20 uL ApaI
20 uL EcoO1091
20 uL ApaI/EcoO1091
2. PCR mixtures and markers (do not heat these samples before loading them into the gel).
a.
Obtain three 0.5-mL tubes. Label them “N,” “R,” and “M.”
b.
Pipet 5 μl SYBR-Green 3X loading buffer into each tube (one pipet tip will do for
this step because you are loading dye into clean tubes).
c.
Using a fresh pipet tip for each addition, place 10 μl of DNA into each tube,
according to its label (“M” is for the Markers, “N” is the PCR mixture missing the
template DNA, and “R” is the PCR mixture complete with template DNA). Mix
the contents by gently aspirating the mixture into the pipet tip and then expelling
the mixture back into the tube. (Be gentle and avoid creating bubbles.)
tube
PCR N
PCR R
M
amount of SYBR-Green
Loading Buffer
5 uL
5 uL
5 uL
amount and identity of sample
PCR with no template DNA
PCR complete with template DNA
markers
BC2004, Spring Semester 2005, Lab Exercise 11-4
BC2004, Spring Semester 2005, Lab Exercise 11-5
C.
Loading the gel.
Load 15
μl of each of your five samples into the wells in your gel, in the following positions.
Load only 6
μl of the BenchTop 1kb DNA Markers/Ladder, “M,” on the chart below.
well number
1 (or 9)
2 (or 10)
3 (or 11)
4 (or 12)
5 (or 13)
6 (or 14)
DNA
sample
PCR “N”
PCR “R”
“M”
Digest A
Digest A/E
Digest E
(*Two pairs of students will load their preparations into one gel. One pair of students will use
lanes 1 through 6, and the other pair of students will use lanes 9 through 14. Cross out the lane
numbers in this table that you and your partner are not using.)
As soon as all twelve preparations have been loaded, notify the instructor. The current should be
started as soon as possible after the wells are loaded; otherwise, the DNA molecules begin to
diffuse into the gel in all directions, making the bands appear fuzzy (you also have to stay in lab
until after the gel has finished running. The sooner you start your gel, the sooner it will be
finished).
+/?
Should you attach the positive or negative lead to this end?
N
R
M
A
A/E
E
1
2
3
4
5
6
long/short?
7
8
N
R
M
A
A/E
E
9
10
11
12
13
14
group 1 lanes
group 2 lanes
Will the longer or shorter pieces of DNA
be found at this end?
gel runs in this direction
long/short?
+/?
Will the longer or shorter pieces of DNA be found at this end?
Should you attach the positive or negative lead to this end?
BC2004, Spring Semester 2005, Lab Exercise 11-6
D.
Electrophoresis
Your instructor will check that there are 12 wells loaded on your gel and will close the apparatus,
attach the electrical leads to the power supply, and switch on the electrical current. The power
supply is regulated to deliver sufficient current to sustain a voltage differential of approximately
120 V across the gel, from cathode to anode. Double check that the gel will be running in the
right direction (it is possible to run the gel in the wrong direction—your samples will run right
off of the top of the gel and into the buffer, from where they cannot be recovered!). Double
check that your gel is running by looking for small bubbles rising up from the electrodes within
the gel apparatus.
SAFETY CONCERN: Please note that 120 V is enough electricity to kill a human being.
Please be very careful with the gel apparatus. It is arranged in such a way that it should be
completely safe if it is used as directed. This is the reason that your instructor will operate the
power supplies (she has experience using the machines safely!)
After approximately 60 minutes, the indicator dye should have traveled approximately 2/3 of the
way down the gel, and the current will be switched off and the electrical leads disconnected.
Demonstration:
The instructor will demonstrate for you the removal of a gel from the apparatus to the
visualization light box. Wearing latex gloves, your instructor will lift the gel tray from the
apparatus with the tray slanting downward at one end, where it will be supported by the
instructor’s gloved fingers. Then, holding the tray over the light box, she will release the gel and
let it slowly slide into the stain solution. Note: If the tray is not slanted toward the end that is
blocked by the instructor’s fingers, the gel is quite likely to slip out the other end of the tray onto
the bench or the floor of the laboratory. This is invariably disappointing.
Recording your data:
You will need to record your data carefully. Place a piece of transparency film over the filter that
is on top of your gel. Carefully outline the gel. Mark the location of your six wells
PRECISELY. Carefully trace the visible bands from the gel onto your transparency. A few
millimeters will make a difference in your calculations, so be sure to perform this step carefully
and precisely. If you have questions, please ask!
To turn in: USE THE INSTRUCTIONS ON THE OFFICIAL LAB HANDOUT, NOT THIS
SUPPLEMENTAL ONE. You are encouraged to work together, but write up all your answers to
the questions in your own words. Your worksheets will not be handed in separately, but will be
handed in as a part of your lab notebook, which is due by 12 noon on Monday, May 2, 2005.
BC2004, Spring Semester 2005, Lab Exercise 11-7
Analyses
A. Construction of the standard curve.
The solution of “Markers” that you loaded into your gel contained DNA molecules that varied in
size from 1,000 to 10,000 bp (base pairs), in increments of 1,000 bp. In your Marker lane, there
should be 10 bands. To construct the standard curve:
1. Measure, in mm (estimate to 0.1 mm), the distance migrated by each of the 10 bands in
the markers lane (it would be best to measure from your gel directly or from a
photograph, but we will measure from our tracings this week in lab). This is why it is so
important that you accurately and precisely trace the data from your gel onto your piece
of transparency film.
Measure from the bottom of the well at the beginning of the lane to the middle of each
band. Record the distances in Data Table A.
2. For each marker band, plot the distance on the X-axis and the molecular size on the Yaxis. Semilogarithmic graph paper will be provided so that as the molecular size of each
band is plotted, the points will appear on the paper in positions that represent their
logarithms; it is not necessary to look up the logarithm of each molecular size. Your
semilogarithmic graph paper will have a linear scale on the X-axis and a logarithmic scale
on the Y-axis. Don’t forget to label your axes and their units (as always!). You may use
Excel if you prefer.
3. Draw a line of best fit through all the points that together clearly generate a straight line.
4. To use this graph to estimate the size of molecules in any other band on the same gel:
i. Measure the distance the band has migrated.
ii. Find its distance on the X-axis, follow a vertical line upward until it intersects with the
line of best fit, then follow a horizontal line toward the left to its intersection with the
Y-axis. The value at which this line intersects the Y-axis is the size of the DNA
molecules in the band. If you make your graph on Excel, you can use the equation for
the line to calculate the molecular sizes.
BC2004, Spring Semester 2005, Lab Exercise 11-8
Data Table A. DNA Markers/Ladder Results (lane 3 or 11)
Band number
Length of markers in
BenchTop 1kb DNA
Ladder (bp)
(begin counting the 1st band
from the top/wells)
Distance from bottom of well to middle of
band, in mm
10,000
8,000
6,000
5,000*
4,000
3,000
2,000
1,000
*Bold: These bands should appear more intense than the others. You might not be able to see all
10 bands. This can make it difficult to know which band is which (and this is important). If you
have any questions, PLEASE ask your instructor! The analyses of your other results depend on
the correct identification of the marker bands.
Important: This diagram is NOT to scale.
10kb 8kb 6kb
5kb…
Markers
wells band 1
2
3
4
5
6…
BC2004, Spring Semester 2005, Lab Exercise 11-9
Agarose Gel Electrophoresis Results: Please tape your transparency tracing (or photograph) of
your gel in the space below. Be sure to clearly label all of your lanes! (Not just lanes 1, 2, 3; but
what is in them.)
BC2004, Spring Semester 2005, Lab Exercise 11-10
Insert your semilogarithmic standard curve here (you may replace this page with your graph or
tape it in the space below). Be sure to label your axes and include units. Include an appropriate
title.
BC2004, Spring Semester 2005, Lab Exercise 11-11
B. Analysis of your PCR product(s).
You prepared two PCR reaction mixtures in Exercise 9 – one labeled “N” because No template
DNA was added to the reaction mixture, and the other labeled “R” because template DNA was
added and a polymerase chain Reaction should have occurred.
Examine the lanes into which DNA from each PCR mixture migrated. There should not be any
detectable product in the “N” mixture; if there is detectable product, the mixture you placed in
the thermal cycler contained unintended template DNA. Because the source and identity of such
DNA are unknown, and such contaminating DNA might also have been present in your “R”
mixture, any detectable PCR product in the “R” lane could be molecules amplified from
contaminating DNA. The preparation would have to be repeated (not, however, within this lab
course).
If there is no detectable product in the “N” lane, but there is a single product band in the “R”
lane, measure the distance and estimate the size of your PCR product. Record these values in
Data Table B.
Data Table B. PCR Products
Use this table to record the distance from the bottom of the wells to the middle of each band in
these lanes. Use your standard curve to estimate the size of each band.
Lane number:
1 (or 9)
2 (or 10)
Sample:
PCR - N
PCR - R
Band number
(from top)
distance (mm)
size (bp)
distance (mm)
size (bp)
BC2004, Spring Semester 2005, Lab Exercise 11-12
C. Analysis of restriction digests: localization of the Apa I target site.
In Exercise 10, you prepared three restriction digestion mixtures of lambda bacteriophage DNA.
The digests loaded into the gel should have contained:
from tube “A”: Apa I - generated restriction fragments,
from tube “E”: EcoO 1091 - generated restriction fragments,
and from tube “A/E”: restriction fragments generated by the simultaneous action of both
enzymes.
Examine the lanes that were loaded with each of the digests, measure the distance migrated by
each of the discernible bands, and estimate the sizes of the molecules in each band from your
standard curve. Record these values in Data Table C.
Consult the diagram provided in the recitation period, noting that:

EcoO 1091 cuts lambda DNA at three sites, yielding four restriction fragments:
2,815 base pairs (bp); 25,982 bp; 19,676 bp; and 29 bp in length.

Apa I cuts lambda DNA at only one site, yielding two restriction fragments:
10,086 bp and 38,416 bp in length.
The goal of this analysis is to locate (to “map”) the single target site of Apa I relative to the target
sites of EcoO 1091. That location can be inferred from the restriction fragment lengths, as
follows.
If the target site of Apa I is located toward the same end of the lambda genome that is
nearer the EcoO 1091 cut that produces the 2,815-bp fragment (the end labeled “A” in the
diagram), then simultaneous digestion with the two enzymes will yield five fragments:
19,676 bp; 18,711 bp; 7,271 bp; 2,815 bp; and 29 bp.
Alternatively:
If the target site of Apa I is located toward the opposite end of the lambda genome
(labeled “B” in the diagram), then simultaneous digestion with the two enzymes will yield
a different set of five fragments:
25,982 bp; 10,057 bp; 9,619 bp; 2,815 bp; and 29 bp.
BC2004, Spring Semester 2005, Lab Exercise 11-13
Unfortunately, gel electrophoretic analysis has its limitations, one of which will require that the
gel be closely and accurately measured. Agarose gels are low-resolution gels, and in each of the
two possible sets of five fragments, there is a pair of bands that will appear as a single band: the
19,676-bp and 18,711-bp bands will not be resolved as two bands, and the 10,057-bp and the
9,619-bp bands will not be resolved as two bands. (These pairs are bracketed on the diagram
provided in recitation for this analysis.) The 29-bp band will move very quickly through the gel,
run off the end into the buffer, and will not be visible at all.
Fortunately, the analysis is still possible, because you will be studying your “A/E” lane to
determine whether the middle of the three visible bands has migrated as molecules of
approximately 10,000 bp, or as molecules of approximately 7,000 bp. Thus, the size of the
molecules in this band will allow you to locate the Apa I site relative to the EcoO I sites.
Data Table C. Restriction Fragments
Use this table to record the distance from the bottom of the wells to the middle of each band in
these lanes. Use your standard curve to estimate the size of each band.
Lane
no.:
4 (or 12)
5 (or 13)
6 (or 14)
Sample:
Digest A
Digest A/E
Digest E
Band no.
distance (mm)
size (bp)
distance (mm)
size (bp)
distance (mm)
size (bp)
BC2004, Spring Semester 2005, Lab Exercise 11-14
Conclusions and thought questions
O. General gel electrophoresis questions
1. Can you see the DNA on your gel under fluorescent room lights? Why or why not?
2. Briefly describe how you visualized the location of DNA bands on your gel.
4. Specifically, physically, of what is a “band” on your gel composed?
5. Could you use agarose gel electrophoresis to quantitate the amount of DNA in a sample? Why or
why not? If not, please describe how you could quantitate the amount of DNA in a sample.
BC2004, Spring Semester 2005, Lab Exercise 11-15
A. Standard Curve
1. Is your standard curve a straight line? Why or why not?
2. Calculate the slope of your line, manually or using Excel. Record your slope in the space below.
3. What are the units for your x-axis? _______ What are the units for your y-axis? _______
4. Briefly explain why you used a linear scale for your x-axis and a logarithmic scale for your yaxis?
5. Briefly explain why you loaded a DNA marker on your gel.
6. Speculate on why DNA markers are also called DNA ladders?
BC2004, Spring Semester 2005, Lab Exercise 11-16
B. PCR
1. Please complete the appropriate number of rows in the following table.
List the ingredients in
tube “N.”
List the ingredients in tube
“R.”
Role of ingredient in PCR
reactions.
2. a. Should you have amplified a product in tube “N”? Why or why not?
b. Did you successfully amplify a product in your tube “N”? If your results did not follow
your prediction, speculate on why.
BC2004, Spring Semester 2005, Lab Exercise 11-17
3. a. Should you have amplified a product in tube “R”? Why or why not?
b. Did you successfully amplify a product in your tube “R”? If your results did not follow
your prediction, speculate on why.
4. What was the size of your PCR product? (Don’t forget to give units!) _________________
5. Briefly explain how you estimated the size of your PCR product.
BC2004, Spring Semester 2005, Lab Exercise 11-18
C. Restriction Digest
1. List the ingredients in your restriction digest reaction tubes and what each ingredient did.
2. Remembering that the Lambda DNA you began with was already in linear form, please
complete the following table with your predictions:
sample
predicted size(s) of restriction digest fragment(s)
uncut
EcoO1091
Apa I
3. Complete the first four lanes on the diagram below with what your gel would look like with
the digest fragments you listed in your answer to question 2.
uncut
EcoO 1091
ApaI
DNA markers
if near end A
if near end B
BC2004, Spring Semester 2005, Lab Exercise 11-19
4. Complete the last two lanes on the diagram above. Draw what you predict you should see if
Apa I cuts near end A or near end B. (These “ends” will be described during recitation.)
5. Which of these two predictions/hypotheses is supported by your data?
6. Draw a restriction map for linearized lambda DNA that is consistent with your data. This
should give the relative locations of the EcoO 1091 and Apa I restriction recognition sites and the
approximate distances (in bp) between the sizes.
7. Given the restriction map below, predict the sizes (in bp) of the restriction digests shown.
1000 bp
BamHI
500 bp
BamHI
HindIII
3000 bp
BamHI
a. If you cut with BamHI, what size fragment(s) will you generate?________________________
b. If you cut with HindIII, what size fragment(s) will you generate? ________________________
c. If you cut with BamHI and HindIII in the same digestion reaction, what size fragment(s) will
you generate?_________________________________________
d. Complete the diagram of the gel from this restriction digest below. Note that you will only
have one band in the HindIII lane (why?).
markers
BamHI
HindIII
BamHI and
HindIII
BC2004, Spring Semester 2005, Lab Exercise 11-20
Materials
Per group of FOUR of students:
1
gel electrophoresis apparatus, mounted with an agarose gel, 0.7 %, covered with
0.5 X Tris-Borate-EDTA (TBE) buffer, pH 8
to prepare one gel:
1
1
0.8 g
100 mL
tape
gel tray
250-mL Erlenmeyer flask
agarose (need scale, weighing paper, and clean spatulas)
0.5 X TBE buffer, pH 8
(to secure one tray)
Per PAIR of students:
DNA solutions from Exercise 9 (PCR-N, PCR-R) and Exercise 10 (Digests A, E and
A/E), thawed and held on ice
55 μl SYBR-Green 3X Gel Loading Buffer
7 μl
DNA size markers (1-kb ladder)
3
1
1
1
1
1
1
1
0.5-ml microfuge tubes
box sterile micropipet tips, for EACH size of micropipettor
micropipettor, 0.5-10 uL
micropipettor, 5-50 uL
transparent mm ruler
gel tray
roll lab tape
gel rig
For class:
1
2-4
4
2
microwave oven
water baths, 65oC
power supplies, electrical lead sets
transilluminators for SYBR-Green
BC2004, Spring Semester 2005, Lab Exercise 11-21
Recipes:
0.5x TBE
Depending on time constraints, it might be wise to purchase 10X TBE.
100ml 10x TBE
1.9L QH2O
Mix and store at room temperature.
0.7% Agarose Gel
In a 250ml Erlenmyer Flask
Add:
0.7g Agarose
100ml 0.5x TBE
Swirl to suspend agarose. Heat uncovered in a microwave oven at the high setting, until
all the agarose is dissolved (~3 minutes). Swirl the solution and check the bottom of the
container to ensure that all the agarose has dissolved (just before complete dissolution,
particles of agarose will appear as translucent grains). Cool until you can hold in your
hand. Carefully pour the agarose solution into the casting tray (with comb in place). Use
a pipet tip to move large bubbles to the sides or end of the tray, while the gel is still
liquid. Allow to solidify.
1M Tris (ph 8.0)
Dissolve:
12.14g
Tris
100ml
QH2O
Adjust pH to 8.0
Dispense into 250ml bottle, label and date.
Loading Dye
already made for 1002 class this semester. If more is needed:
1.5 ml Glycerol
3.5 ml 0.04% Bromophenol Blue, stored on shelf
15 ul SYBR Green (Sigma S9430)
Divide into four tubes to distribute during class for easy student access. Wrap with foil
and store in freezer until use.
TBE (10x) – 0.89M Tris-borate, 0.02M EDTA, pH 8.3
1g
NaOH (m.w. 40.00)
107.8g
Tris Base (m.w. 121.14)
55g
Boric Acid (m.w. 61.83)
7.4g
EDTA (m.w. 372.24)
1.
2.
3.
4.
Add all dry ingredients to 700ml of QH2O in a 2L beaker.
Stir (using magnetic stirrer) to dissolve.
Make sure pH is 8.3. Adjust if necessary.
Add QH2O to bring the total solution to 1L.
BC2004, Spring Semester 2005, Lab Exercise 11-22