Molecular Biology
Lab
M116L
Spring 2015
Table of Contents
SAFETY AND LAB CITIZENSHIP ................................................................................................................................ 1
LAB 1 ..................................................................................................................................................................... 2
1A. USE OF MICROPIPETTORS ...........................................................................................................................................2
General Use of Micropipettors: .............................................................................................................................3
Fun with Pipettors .................................................................................................................................................3
1B. IDENTIFICATION OF YOUR D1S80 GENOTYPE .................................................................................................................4
Protocol – Isolation of cheek cells .........................................................................................................................5
Protocol - PCR Procedure ......................................................................................................................................6
LAB 2 ..................................................................................................................................................................... 7
2A. RESULTS OF THE IDENTIFICATION OF YOUR D1S80 GENOTYPE ...........................................................................................7
2B. GEL ELECTROPHORESIS ..............................................................................................................................................7
Protocol - Gel electrophoresis ...............................................................................................................................8
2C. EXPERIMENTAL DESIGN: INVESTIGATION OF VARIABLES AFFECTING RESTRICTION ENZYME DIGESTS.............................................9
LAB 3 ................................................................................................................................................................... 12
EFFECTS OF DIFFERENT VARIABLES ON THE ACTIVITY OF RES. .................................................................................................12
LAB 4 ................................................................................................................................................................... 13
METHYLATION OF DNA.................................................................................................................................................13
Protocol – Checking for methylase activity .........................................................................................................14
LAB 5 ................................................................................................................................................................... 15
5A. DIGESTION OF PLASMID DNA...................................................................................................................................15
Protocol – RE digestion of plasmid ......................................................................................................................16
5B. TRANSFORMATION .................................................................................................................................................16
Protocol - Transformation ...................................................................................................................................17
LAB 6 ................................................................................................................................................................... 19
6A. ISOLATION OF PAR 1.5 PLASMID FROM SUCCESSFULLY TRANSFORMED CELLS......................................................................19
Protocol – Qiagen plasmid miniprep ...................................................................................................................19
6B. RESTRICTION MAPPING OF PLASMID DNA ..................................................................................................................20
Protocol – RE digestion of plasmid ......................................................................................................................21
LAB 7 ................................................................................................................................................................... 22
7A: PICKING A CANDIDATE GENE .....................................................................................................................................24
7B: DESIGNING PRIMERS TO TEST YOUR CANDIDATE GENE ....................................................................................................25
LAB 8 ................................................................................................................................................................... 29
8A: ISOLATION OF TOTAL RNA FROM CELLS ......................................................................................................................29
Protocol – RNA isolation .....................................................................................................................................30
8B: MAKING CDNA FROM TOTAL RNA ............................................................................................................................31
Protocol – cDNA synthesis...................................................................................................................................32
8C: QUANTIFYING GENE EXPRESSION USING QPCR ............................................................................................................32
Protocol - qPCR ...................................................................................................................................................34
USING YOUR QPCR RESULTS (CT METHOD) ...................................................................................................... 36
SUPPORTING INFORMATION ............................................................................................................................... 37
EXAMPLE RESTRICTION ENZYME INFORMATION..................................................................................................................38
DNA LADDERS USED IN EXPERIMENTS ..............................................................................................................................42
PBK-CMV INFORMATION .............................................................................................................................................. 45
Safety and lab citizenship
THINK ABOUT EVERYTHING YOU ARE DOING, and judiciously use your common sense. Be aware that the lab is
fraught with potential hazards, and know that you are just one stupid accident away from causing yourself or others
around you serious injury. You will be advised when chemicals you are working with are hazardous, either by your
instructor, or as noted in the lab manual. PAY ATTENTION to these warnings, and be extra careful when working
with these reagents. Working in lab is NOT like driving:
 You cannot text.

You cannot call your BFF.

You cannot invite your BFF (or anyone else!) to come with you to lab; entrance into the lab is at the
discretion of the TA\Instructor\Lab coordinator.

You cannot eat, smoke, put on makeup or drink in lab.

You cannot be drunk or high on any kind of drug in lab. Not even medicinal marijuana. You can be high on
life.

You MUST wear closed-toed shoes in lab.

You must wear your lab coat AT ALL TIMES in lab.

DO NOT mouth pipette in lab.

Clean your bench and common areas before you leave.

If you make a mess, clean it up. If you are reported creating a mess and NOT cleaning it up, after one
warning, you will be penalized 10% of your final score. There are other people working in lab, and other
sections using the lab, and it is EVERYONE'S responsibility to maintain the lab and equipment.

If you break a piece of equipment (or notice that something is not working), report it immediately.
Cleaning up

Remove all labeling tape from glass and plastic ware after your experiment.

Remove all writing from glass and plastic ware after your experiment.

Place dirty labware in the tub labeled (Duh!) "DIRTY LABWARE".

Microfuge tubes and micropipette tips should be disposed of in the red bag in the large red plastic container
in the front of the room.

Flasks with bacterial cultures are to be placed in the tub marked "CONTAMINATED LABWARE".

Ethidium bromide containing solutions, and any other waste solutions, must be disposed of in the liquid
waste barrel in the back of the room (near the refrigerator).

Gels should be thrown away in the gel bucket (next to the UV illuminator).

Swab your bench clean, and put away all equipment.

DO NOT dispose of any solutions in the sink without first checking with your instructor.
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Lab 1
1A. Use of micropipettors
Methods in molecular biology involve extensive use of a variety of specialized but fairly simple equipment. Most
reactions in manipulating nucleic acids are performed in small microfuge tubes in volumes as small as 10µl. In order
to successfully perform the experiments in this manual, you must be able to dispense volumes as small as 0.5µl.
Digital micropipettors are used to accurately dispense volumes less than 1 milliliter (ml). Various sized micropipettors
are used, depending on the volume to be transferred.
Micropipettor
Range of accuracy
Viewing window (top to bottom)
Example
P-20
1 – 20 µL
0-8-7 = 8.7 µL
P-200
20 – 200 µL
1-1-5 = 115 µL
P-1000
200 – 1000 µL
0-8-7 = 870 µL
CAUTION: Micropipettors are expensive and can be easily damaged if not handled properly. Always take the
following precautions when using the pipettors:

Never rotate the volume adjuster beyond the upper or lower limits of the pipet.

Never use the pipettor without an appropriate disposable tip in place.

Never invert or lay the pipettor down with a filled tip.

Always hold the pipettor with the tip pointing down, never horizontal or up. Fluid could run back into the
piston and damage it or contaminate your next reagent.

Never let the plunger snap back after withdrawing or expelling fluid.

Always slowly release the plunger.

Never put the barrel in fluid.

Only put the very end of the disposable tip into the fluid.

Never flame the barrel or the tip of a pipettor. The pipet tips are pre-sterilized by autoclaving and do not
need to be flamed.

Always use a new tip for each new reagent.
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General Use of Micropipettors:
1. Rotate the volume adjuster knob to desired setting.
Remember: DO NOT try to adjust outside of the range for the micropipettor.
2. Attach a yellow or white pipet tip to the end of the pipettor. Hold the base of the pipet tip box and press
firmly with a slight twist to ensure an airtight seal.
Most micropipettors have a 2 position plunger. Depressing to the first stop measures the desired
volume. Go only to this first stop when taking up the solution. Depressing to the second stop
pushes a volume of air through the tip to expel any solution remaining in it. Go to this second stop
when dispensing the solution.
3. Depress the plunger to the first stop. Hold the pipettor vertically and immerse the tip 1-2 mm into the
solution to be pipetted. Slowly release the plunger to draw fluid into the tip. If the plunger is depressed after
the tip is in the solution, a tiny air bubble is created which will prevent the proper volume from being
pipetted.
4. Remove any droplets stuck to the outside of the tip by sliding the pipet tip along the inside wall of the tube.
5. To expel the sample, place the tip against the inside wall of the reaction tube. Slowly depress the plunger to
the first stop. Then depress to the second stop to expel any fluid remaining in the tip.
6. With the plunger fully depressed, slide the pipettor from the tube. Do not release the plunger until the pipet
tip is out of the tube.
7. Discard the tip by depressing the ejector button or remove it manually.
Fun with Pipettors
1. Label 3 1.5 ml microfuge tubes "A", "B", & "C".
2. Use the table below as a checklist for the addition of solutions to each tube.
3. Set the small-volume pipettor (P-20) to 4µl volume and add solution 1 to each tube. Then using a fresh tip
each time, add the appropriate volume of each remaining solution to each tube.
4. Close the tops. Mix the reagents by tapping the tube several times. Pool the reagents by:
a) sharply tapping the tube bottom on the bench top; or
b) briefly spinning the tubes in the microfuge
5. Check the accuracy of your measurements. Each microfuge tube should contain 10ul. Set the pipettor to
10µl and carefully withdraw the solution from each tube.
Does the fluid fill the tip? Is there an air space left at the end of the tip? Is there any fluid remaining in the tube? If any
measurement was inaccurate, repeat the exercise.
If you are using a P-200, follow the above instructions and add 10 times the amount.
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1B. Identification of your D1S80 genotype
The field of molecular biology has been driven by the development of theories and procedures that revolutionize the
way scientists think about and address problems. DNA cloning, Southern blotting, and DNA sequencing are but a few
examples. The polymerase chain reaction (PCR), developed at the Cetus Corporation by Kary Mullis is another
example of how a relatively simple procedure is having revolutionary impact in all areas of life science. Based on the
significance of his contribution, Kary Mullis was awarded the 1993 Nobel Prize in Chemistry for the development of
PCR.
The polymerase chain reaction (PCR) is a rapid in vitro method for enzymatic synthesis of a specific DNA sequence.
Microgram quantities of DNA can be produced from picogram amounts of starting material. The procedure is simple
in concept, yet elegant in overall design. Two oligonucleotides are used as primers for a series of reactions catalyzed
by a DNA polymerase. The oligo primers typically have different sequences complementary to sequences that lie on
opposite strands of the template and lank the segment of DNA to be amplified. Template (or target) DNA is
denatured and then cooled to a temperature that allows the oligo primers to anneal to their target sequences and
provide the 3'-OH required for DNA synthesis. The primers are then extended with DNA polymerase, synthesizing a
new DNA strand complementary to the target. Denaturation, annealing, and DNA synthesis are then repeated many
times. The products of one round of amplification serve as templates for the next round, doubling the amount of DNA
product with each cycle.
The polymerase used in PCR is produced by the thermophilic bacterium, Thermus aquaticus. This enzyme can
survive extended periods at 95°C, is not deactivated by the heat denaturation step and does not need to be replaced
at each round of the amplification cycle. In addition, extension can take place at 75°C which minimizes mismatch
extensions. The newly synthesized strand initially extends a variable distance beyond the other primer-binding site on
the complementary strand, creating additional primer-binding sites on the newly synthesized strand. In subsequent
cycles, the primers are extended only to the binding site of the second primer, resulting in an amplified product
defined by the two primers. The original and variable-length strands persist in the reaction, but variable-length
strands increase at an arithmetic rate, while the primer-defined length strands increase exponentially. Thus, after 2030 cycles, virtually all of the product consists of the primer-defined length strands. This exponential accumulation of
DNA sequences can theoretically produce over a million fold amplification of the target in just 20 cycles. In actual
practice, the amplification efficiency is less than 100% and 30-40 cycles are commonly done.
New applications of the PCR are being developed constantly. This procedure can be used to amplify and clone
extremely rare sequences from the large genomes of eukaryotes, screen phage and plasmid libraries, produce
templates for DNA sequencing, produce large quantities of probe DNA, diagnose infectious and genetic diseases,
amplify minute quantities of DNA at crime scenes for forensic analysis, and create fingerprint-like patterns of
amplified DNA for the identification of individuals and paternity testing. The applications are virtually endless.
The basic PCR protocol includes three steps; denaturation, annealing, and extension. In the initial denaturation step,
the DNA template is melted by incubation at 95°C for 1-3 minutes. Subsequent denaturation is carried out at 95°C
for 30 seconds. Primers should be 18-30 nucleotides in length, 50-60% G+C, and if possible, have the same Tms.
The optimal annealing temperature is 5°C below the Tm. During the extension step, Taq DNA polymerase extends
from the 3' ends of the annealed primers. The rate of extension at 72°C has been estimated to vary from 35 to 100
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nucleotides per second depending on the buffer, pH, salt concentration, and DNA template. One minute should be
long enough to produce products up to 2 kilobases in length.
In this exercise, the PCR is used to amplify a short nucleotide sequence from chromosome 1. Many regions of
human chromosomes exhibit a great deal of diversity. The evolutionary principle of variation within a population is a
cornerstone in biology. This variation results from subtle differences in the DNA sequence in individuals of a given
species, and the DNA of Homo sapiens is no exception. Variation commonly originates by the mistaken duplication of
a small sequence of nucleotides when only one copy was present before replication. This results in a tandem repeat
of the original sequence. If this mistake occurs again in another round of replication, then three copies of a sequence
will be in tandem. These tandem repeats are part of our chromosomes and as such, they will be inherited according
to Mendelian genetics. These regions are termed polymorphic and are usually found in DNA that does not code for
protein. Over the centuries, the number of tandem repeat units has increased, therefore each of us has inherited a
variable number of tandem repeats (VNTR) at many loci scattered throughout our genomes. A VNTR can be thought
of as a locus with each particular number of repeated units being analogous to different alleles. Therefore, each
human (except for identical twins) carries a unique combination of VNTR's and these alleles can be used in
population studies or to identify a particular individual.
PCR can be used to amplify portions of human DNA that are known to contain VNTR's. The repeats have no known
relationship to any disease state, sex determination, or any other human phenotype, but can be used in forensic
casework to help discriminate between DNA samples from different individuals. So far, 29 alleles have been
identified, ranging in size from about 350 to 1000 bp and determine as many as 435 possible genotypes.
The VNTR (D1S80) amplified in this lab exercise is located in a non-coding region of chromosome I and has a core
size of 142 bp. Every repeat unit will add 16 base pairs to the VNTR. You will isolate some of your own cells (cheek
cells), extract DNA from them to use as the template for the PCR, amplify the D1S80 region, and run the PCR
products out on an agarose gel. You will use the estimated molecular weights of each band to determine how many
repeat units are in each allele. The following formula should be used:
Tandem repeats=
Observed band size-142
16
Protocol – Isolation of cheek cells
This is a painless, bloodless, and noninvasive method for cell collection. The risk of spreading an infectious
agent by this procedure is less likely than natural processes such as coughing and sneezing. Everyone will
participate in this experiment.
1. Label a sterile centrifuge tube with your initials.
2. Pour all the saline solution from materials supplied into your mouth and vigorously swish it around for 30
sec. It is a good idea not to eat immediately prior to this isolation, but food particles should have little effect
on the results.
3. Expel the solution into the labeled centrifuge tube (DO NOT TAPE).
4. Close the cap securely and spin at 3000rpm for 10min to pellet the cells.
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5. Without disturbing the pellet, use a micropipette to take off as much supernatant as possible. Place the tube
on ice. If the pellet does not adhere well to the tube, pour off as much supernatant as possible and then
pipet off the rest.
6.
Add 500µl of the 10% Chelex solution to the pellet. To do this, pipette Chelex beads up and down several
times, and before the beads settle, transfer 500µl to the microfuge tube with the pellet.
Chelex is a resin which binds heavy metal ions that inhibit PCR.
7. Resuspend the cells in the Chelex by pipetting up and down; make certain that no visible cell clumps
remain.
8. Transfer 500µl of the Chelex-cell sample to a labeled microfuge tube. Incubate in the heating block for 15
minutes at 100°C. USE FORCEPS TO PLACE THE TUBE INTO AND TO REMOVE IT FROM THE
HEATING BLOCK.
9. Remove tube from block and cool on ice for 1 minute.
Boiling lyses the cells, releasing the nuclear contents.
10. Spin the tube in the microfuge for 30 sec to pellet the Chelex beads and cell debris.
11. Carefully transfer 200µl of the supernatant which contains chromosomal DNA to a fresh microfuge tube and
place it on ice. Do not transfer any of the Chelex beads.
Protocol - PCR Procedure
PCR is very sensitive. Wear gloves to help prevent contamination.
1. Label a 0.2ml PCR tube with your initials (DO NOT USE TAPE).
2. Add the reagents in the following order.
10µl
1µl
1µl
45µl
Cheek cell DNA
us oligo
ds oligo
PCR mix1
Mix and pool. Save the rest of your unamplified cheek cell DNA. Keep your PCR cocktail on ice until ready
to start the PCR procedure.
3. Place in PCR tray, when all reaction mixes are ready, place the tray in the PCR machine. Press "Go" twice.
When the program has been completed, your tubes will be removed and refrigerated
until the next lab period, when you will run an agarose gel to analyze the PCR
products.
1
PCR mix: 2.5U Taq DNA polymerase, 0.25mM dNTPs, 0.25mM MgCI2
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Lab 2
2A. Results of the identification of your D1S80 genotype
In this exercise, we will compare a variety of D1S80 alleles found among the students in the class.
1. Transfer 20µl of the amplification product from your PCR reaction tube into a clean microfuge tube.
2. Add 2µl of load dye to your sample, mix and pool.
3. Load 15µl of your sample into ONE sample well in each of the agarose gels provided (3.0%, 1.5% and
0.75%). Note your lane position in each of gels.
One person will load the unamplified control and size markers onto each gel.
4. Run at 100V for 60 min, view, and photograph.
Following electrophoresis and staining, different alleles appear as distinct bands in the gel. The distance migrated is
inversely proportional to the number of repeat units. One or two bands will be visible in each sample lane.
What does this indicate? Are the results different in each gel?
2B. Gel Electrophoresis
Gel electrophoresis through agarose or polyacrylamide is a powerful method for separating mixtures of nucleic acid
molecules. Polyacrylamide can be used for separating both nucleic acids and proteins. It allows very good resolution.
However, it is very difficult to pour and it is a neurotoxin. We won’t be using it in this lab. Agarose is a polysaccharide
isolated from kelp (seaweed). It can be melted in hot buffer and when cooled, forms a semi-solid gel, creating a
mesh-like sieve. Double stranded DNA has a uniform negative charge distribution due to its sugar-phosphate
backbone. Because of this charge, DNA will migrate in an electric field from the negative pole (cathode) to the
positive pole (anode). The mobility of a DNA fragment depends on its molecular weight and is independent of its
sequence. A semi-log plot of fragment size vs. distance migrated will give a straight line. When DNA fragments of
known molecular weight are electrophoresed on the same gel as unknown samples, the standards can be used to
generate a size curve to determine the size of the unknowns. The fractionated molecules can be viewed directly in
the gel and can even be recovered and subjected to subsequent experiments.
Agarose gels can be used to separate fragments between 70 bp and 800,000 bp depending on the percentage of
agarose used. The following table will give you an idea of the range of fragments which can be separated with
agarose gels of different % agarose.
Percent agarose
0.6%
0.8%
1.0%
1.2%
1.5%
Size of fragments
1.0 - 20 kb
0.6 - 12 kb
0.4 - 8 kb
0.3 - 6 kb
0.2 - 4 kb
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Protocol - Gel electrophoresis
In Advance:
1. Add 0.5g agarose to 55ml TAE buffer2 and Microwave the mix on high until the solution boils (about 1.5
min). Swirl and reheat until no clumps remain in the solution. You will pour the gel when the agarose is at
~50°C. You can estimate the temperature of the agarose by holding the beaker on your bare palm. When it
reaches a temperature that is just slightly above comfortable to hold, the agarose is at ~50°C and can be
poured.
2. Put the comb in place and pour the molten agarose into the casting tray. Allow the gel to solidify. The gel will
become translucent as it solidifies.
3. Remove the reaction tubes from the water bath.
4. Add 1µl load dye3 to each tube. (Fresh tips!).
5. Mix and pool.
The load dye serves three purposes: glycerol increases the density of the sample, ensuring that the DNA
drops into the well; bromophenol blue and xylene cyanol are dyes that add color to the sample to make
loading easier; and the dyes, in an electric field, move toward the anode at a predictable rate. This allows us
to determine how far a gel should be run to obtain the desired results. Bromophenol blue migrates about 2.2
times faster than xylene cyanol independent of the agarose concentration over a range of 0.5% to 1.4%.
6.
Load 10µl from each tube into a separate well in the gel. Use a fresh tip for each reaction. Center the pipet
tip over the well. Use both hands to steady the pipettor. Dip the tip into the well just enough to break the
surface of the buffer. Do not insert the tip too far; the well can be punctured by it. Gently depress the plunger
to slowly expel the sample into the well. Carefully remove the pipet tip from the well.
Also load the 100bp DNA Ladder a separate well.
DNA size markers are generated from restriction digests of well-characterized plasmids or bacteriophage (see
appendix). The sizes of the cleavage products of these digests are known and can be used to estimate the size of
unknown DNA fragments by establishing a size curve on semi-log graph paper.
7. Close the top of the gel box. Connect the electrical leads to the power supply-anode to anode and cathode
to cathode. Turn on the power supply and set it to 100 volts.
Gas bubbles rising from the electrodes are the first sign that current is flowing through the system. After a few minutes,
colored bands should be seen moving into the gel in the direction of the positive pole. The rate of migration through the
gel is dependent on the voltage- the higher the voltage the faster the DNA migrates. However, high voltage can cause
problems in minigels. Imperfections in the gel, such as differences in thickness and density, are accentuated by high
voltage and can cause slanted or U-shaped bands. Also the heat generated by high voltage can melt the agarose and
change its sieving properties.
8. Run the gel until the lead color band migrates 2/3 of gel length.
9. Turn off the power supply, disconnect the leads, and remove the top of the gel box.
2
TAE buffer (10X) (For 1 liter): 48.4g Tris base, 11.42ml glacial acetic acid and 20ml 0.5MEDTA pH 8.0
3
10X load dye consists of: 50% glycerol, 0.25% bromophenol blue and 0.25% xylene cyanol in TAE buffer.
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Ethidium bromide (EtBr) has been incorporated into running buffer used during electrophoresis.
REVIEW THE FOLLOWING SECTION ON ETHIDIUM BROMIDE BEFORE
PROCEEDING. WEAR GLOVES THROUGHOUT THE PROCEDURE!
Ethidium bromide is a rapid, sensitive, and reproducible method for staining DNA. However, it requires care in
handling. Always wear gloves when working with ethidium bromide solutions or stained gels. After staining, pour the
ethidium bromide solution into the appropriate waste container. Never pour it down the sink. Ethidium bromide is a
mutagen. It reacts with DNA by intercalating between the stacked bases and will do so to your DNA if there is
contact.
What may result from this?
1. Carefully remove the gel from the electrophoresis box and place it on the UV box.
2. View under a UV light source. Photograph.
DNA stained with EtBr can be seen with ultraviolet light. A mid-wavelength (260-360nm) UV lamp emits light in the
optimum range for illuminating stained gels. The fluorescence of ethidium bromide-DNA complexes emits in the red
orange wavelength range and is ten times greater than that of ethidium bromide alone.
However, UV light can damage both the retina and cornea of the eye; it is a major factor in cataract
formation. Never look directly at an unshielded UV light source. View gels only through a filter or
safety glasses that absorb the harmful wavelengths.
3. Calculate the size of the DNA fragments.
Measure the distance of migration of each marker fragment from front edge of the well to the leading edge of each
band. Graph the size of the fragment (y-axis) against the distance migrated in mm (x-axis) on semi-log paper. The
line of best fit should be nearly straight. To determine the size of the VNTRs, measure the distance of migration for
each one. Locate the distance on the x-axis and extrapolate back to the estimated size. (Draw a vertical line from the
x-axis to the marker data line, then extend a horizontal line to the y-axis).
2C. Experimental design: Investigation of variables affecting restriction
enzyme digests
Technical developments often lead to quantum jumps forward in science. The discovery of two naturally occurring
and quite remarkable classes of biological molecules made possible the development of methods to isolate and
manipulate specific DNA fragments. These two classes of molecules are plasmid DNA and restriction enzymes. We
will first look at restriction enzymes (abbreviated RE).
Restriction enzymes recognize specific sequences in double stranded DNA and cut both strands at specific sites.
They are found in a wide variety of prokaryotes and their biological role is to cleave foreign DNA molecules. The
cell's own DNA is not affected because the sites recognized by its own restriction enzymes are modified (by
methylation) and insensitive to cleavage. The recognition sites are generally 4-8 base pairs in length and exhibit a
twofold rotational symmetry. In other words, the sites are palindromes reading the same in the 5'->3' direction on
both strands. The cleavage sites are positioned symmetrically as well. Cleavage of the two strands can be staggered
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resulting in either a 3’ overhang or a 5' overhang or it can occur at the center position on both strands resulting in
blunt ends. Restriction enzyme cuts that result in 3' or 5' overhangs generate fragment ends ("sticky ends”) that can
be reattached to any other ends generated by the same restriction enzyme. This allows for the dissection, analysis,
and restriction of DNA in a controlled manner.
Hundreds of restriction enzymes have been isolated and characterized thus far. Their names consist of a 3 letter
abbreviation for the host organism followed by a strain designation, if necessary, and a roman numeral indicating the
order of its identification. (For example, EcoRI isolated from Escherichia coli, PvuII from Proteus vulgaris, HindIII from
Hemophilus influenzae, TaqI from Thermus aquaticus) There are three types of restriction enzymes. Type II
restriction enzymes are the most useful for our purposes. These are used by molecular biologists to cut DNA
molecules into specific fragments that can be analyzed and manipulated more easily than the original molecule.
The standard measure of restriction enzyme activity is the unit. This is generally defined as the amount of enzyme
needed to completely digest 1µg of DNA in 1 hour at 37°C. Many restriction enzymes are supplied by
manufacturers in concentrated form; these will be diluted immediately prior to use in 10X restriction enzyme buffer, if
necessary. Optimal buffer conditions vary widely for different enzymes. Commercial enzyme manufacturers supply
the appropriate buffer (usually at 10X) with each enzyme. The enzymes are stored in 50% glycerol (glycerol prevents
the freezing of the enzyme at -20°C) which can inhibit the reaction. Therefore make certain that the amount of stock
restriction enzyme added makes up less than 10% of the final reaction volume. A few other important points
regarding the use of restriction enzymes are:
 Restriction enzymes are most stable at cold temperatures and will lose activity if warmed for any length of
time. Always keep restriction enzymes on ice when in use and in the freezer when not in use.
 Hold the upper part of the tube so heat from your fingers does not affect the enzyme. Do not touch the lip of
the enzyme tube.
 Always use a new pipet tip.
Three important variables that can be used to regulate the activity of REs are time, buffer and enzyme
concentration. Pick one variable and with the help of your TA, design an experimental protocol to test the effects of
this variable on the activity of REs. Refer to the protocol below for an example of an experiment that tests the effect
of temperature on RE activity.
Example protocol to test effect of temperature on RE activity:
Tube
DNA (µl)
10X Buffer
(µL)
RE (µL)
Water (µL)
Incubation
temp. (°C)
1
1.0
1.0
-
8.0
25
2
1.0
1.0
1.0
7.0
25
3
1.0
1.0
-
8.0
30
4
1.0
1.0
1.0
7.0
30
5
1.0
1.0
-
8.0
37
6
1.0
1.0
1.0
7.0
37
1. Set up 6 tubes are shown in the table.
2. Add components and incubate for 60 mins. at the temperature indicated.
3. Remove the tubes and run the reactions on an agarose gel.
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While designing your experiment, keep in mind the following points:
 The components added to an RE reaction are DNA sample, RE buffer and RE. The total volume of the
reaction will be 10µL, which is made up by adding the appropriate volume of water.
 What will you use as negative and positive controls? And why do you need negative and positive controls?
 How will you set up the experiment so that you are only varying the ONE variable that you are going to test?
 What are your predictions for the effect of your chosen variable on RE activity? Would you expect all REs to
respond to the differences in the variable uniformly?
Make a chart/table and write out a protocol that you will use in the next lab to test the
effect of your variable of interest on RE activity. Compare your table/protocol to other
groups that are testing the same variable. Make any changes you deem necessary,
and come up with the final procedure that you will use for next class.
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Lab 3
Effects of different variables on the activity of REs.
Set up your experiment to test the effect of a variable on RE activity based on the protocol that you came up with in
the last class.
Once you have completed the activity assay, run your reactions on an agarose gel to get your results. Refer to Lab
2B on details on how to run a gel.
When you are setting up your experiment, be sure to note down the following, so you can use the information to
interpret your results:
What was the sample of DNA that was digested?
What was the concentration of the DNA?
What enzyme did you use?
What the concentration of the enzyme you used?
Pg| 12
Lab 4
Methylation of DNA
Restriction endonucleases protect the bacterial cell by cutting foreign DNA. For example, bacteriophage DNA may
invade a bacterial cell, and a restriction enzyme made by the bacteria will degrade this foreign DNA, but leave the
bacterial DNA untouched. The reason for this is that within the bacterial DNA where there is a recognition site for the
restriction enzyme, nucleotides in that site are chemically modified to prevent the restriction enzyme from acting at
that site. For many type II restriction enzymes, there is a corresponding methylase that modifies the recognition
sequence and renders it resistant to cleavage. As a result, unmodified foreign DNA (such as bacteriophage DNA)
that enters a host cell is rapidly degraded and the modified host DNA is untouched.
Methylation of DNA also occurs in organisms other than bacteria. In mammalian DNA, the degree of methylation of
cytosines has been correlated with gene activity. C-5 of cytosine can be methylated by specific methyltransferases,
most often on a cytosine immediately 5' of a guanine residue. About seventy percent of CpG sequences in
mammalian DNA are estimated to be methylated. Studies using restriction enzymes sensitive to methylation have
shown that inactive genes are more extensively methylated than active ones. Different tissues from the same
individual consistently have a cell-type-specific pattern of sequences containing 5-methyl-C. For example, a liver
protein gene is more methylated in the brain because it is inactivated. It has been proposed that the protruding
methyl group interferes with the binding of a factor necessary for transcription.
In this lab, we will attempt to figure out the specificity of methylases. Just like REs, methylases recognize specific
sequences of DNA and methylate at a specific residue within that sequence. For example, the methylase M.EcoRI
adds a methyl group to the second A in this recognition site (GAATTC). This particular methylase is specific for this
recognition site, just as the EcoRI restriction enzyme is specific for the same sequence. This addition protects the
recognition site from cleavage by EcoRI. The exact mechanism by which methylation confers resistance to RE
digestion is not known. It is possible that the RE no longer recognizes the sequence, or if it does, cannot bind to the
sequence due to the methyl group.
This lab will use lambda () DNA, which is a 48.5 kb phage DNA with 5 EcoRI recognition sites. We will test whether
the methylase we use is specific to EcoRI, HindIII, both, or neither. The 2X buffer is specific for the methylase and
contains S-adenosyl methionine (SAM), which acts as the methyl group donor. After the methylation reaction, the
tube contents are heated to inactivate the methylase. Then an aliquot of buffer is added (to compensate for the
higher salt concentration required by EcoRI), as well as the restriction enzyme.
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Protocol – Checking for methylase activity
1. Label 6 microfuge tubes as shown in the table below.
2. Use the following table as a checklist for the addition of reagents to the tubes.
Tube
DNA
2X SAM Buffer
Methylase
Water
M-E-
4 µL
5 µL
--
1 µL
M+E-
4 µL
5 µL
1 µL
--
M-E+
4 µL
5 µL
--
1 µL
M+E+
4 µL
5 µL
1 µL
--
M-H+
4 µL
5 µL
--
1 µL
M+H+
4 µL
5 µL
1 µL
--
3. Add the reagents to each tube4. Mix and pool. Incubate the reactions in the 37°C water bath for 45 min.
4. Remove the tubes from the bath.
5. Incubate the samples at 65°C for 20 min.
6. Use the table below as checklist for the addition of the remaining reagents.
Tube
2XKGB EcoRI
HinDIII
M-E-
10 µL
--
--
M+E-
10 µL
--
--
M-E+
9 µL
1 µL
--
M+E+
9 µL
1 µL
--
M-H+
9 µL
--
1 µL
M+H+
9 µL
--
1 µL
7. Mix and pool. Incubate all the tubes once again at 37°C for 45 min.
8. Remove the tubes from the water bath. Add 2µl load dye to each tube.
9. Load 10µl from each reaction onto a 1.0% agarose gel. Run at 100V for 40-60'.
10. Stain, view, and photograph. (Review safety precautions from previous lab).
4
2XSAM bufer: 5µl SAM, 100µl 10X bufer, 370µl dH2O, 25µl BSA
Pg| 14
Lab 5
The overall goal is to give you an idea of how genes of interest are “cloned” into vectors. In this set of labs, we will
first generate the baseline plasmid map of pBK-CMV. We will then “transform” bacterial cells with a variant of this
plasmid containing a gene inserted into it. We will then isolate the plasmid containing the insert from the successfully
transformed cells, and generate a map of this plasmid, and compare it with the baseline plasmid map of just the
pBK-CMV to derive information about our “clone”.
5A. Digestion of Plasmid DNA
Plasmids are usually double stranded, closed-circular DNA molecules which occur naturally in most species of
bacteria. They are generally not essential to the cell under all or most conditions but can be selected for and
maintained under certain conditions, based on the genetic information they carry. Plasmids range in size from two to
several hundred kilobases, are self-replicating, and do not integrate into the host genome. Plasmids now used by
modem molecular biologists are not usually naturally occurring ones; they are generally derived from several sources
using recombinant DNA techniques to carry very specific traits and are known as vectors. All plasmid vectors must
have three features to be of use: at least one selectable marker, an origin of replication, and a unique restriction
enzyme cutting site (most often a multiple cloning site).
The selectable marker is often a gene which confers resistance to an antibiotic. Other selectable markers may
involve introduction of a gene encoding a specific enzyme allowing a new metabolic function in the recipient cells.
Bacterial cells containing the plasmid will survive and grow in media containing the antibiotic, or substrate for the new
enzyme. The origin of replication, or ori site, allows the plasmid to replicate as extra chromosomal DNA, independent
of the bacterial chromosome. The unique restriction enzyme cutting site, or cloning site, is necessary for the insertion
of the DNA of interest into the plasmid. The multiple cloning sites usually consist of a sequence containing a number
of restriction enzyme cleavage sites adjacent to one another and present only once in the plasmid.
The plasmid used in this exercise is pBK-CMV, a state-of-the-art synthetic vector developed by Stratagene. It
contains several features useful for molecular biology. The multiple cloning site contains 17 unique restriction
enzyme sites. There is a neomycin resistance gene which allows for selection in both prokaryotes and eukaryotes.
Prokaryotic expression is driven by the lac promoter and eukaryotic expression by the cytomegalovirus (CMV)
immediate early promoter; the protein of interest can therefore be produced in both prokaryotes and eukaryotes.
Pg| 15
Protocol – RE digestion of plasmid
1. Label 7 tubes – "S","E","P", "-", "S+E", "S+P", "E+P".
2. Use the tubes as a checklist for addition of reagents.
Tube
DNA
S
E
P
-S+P
S+E
E+P
5 µL
5 µL
5 µL
5 µL
5 µL
5 µL
5 µL
10X Buffer
1 µL A
1 µL H
1 µL H
1 µL H
1 µL A
1 µL A
1 µL H
SacI
EcoRI
1 µL
---1 µL
1 µL
--
-1 µL
---1 µL
1 µL
PstI
--1 µL
-1 µL
-1 µL
Water
3 µL
3 µL
3 µL
4 µL
2 µL
2 µL
2 µL
3. Use fresh tips for each new reagent. Add the enzyme last.
4.
Mix and pool. Place the tubes in 37°C water bath for 60min.
The buffer used depends on the optimal assay conditions for each enzyme. These conditions are determined by the manufacturer of
the enzyme. The buffers usually contain Tris, Mg2+ and the proper salt concentration to give optimal results.
What sequences do the restriction enzymes recognize?
Where are the cuts made?
What type of ends are generated by each?
5. Add 1µl load dye to each reaction. Load onto a 1% gel, run at 100V for 60 or more minutes. Also load
markers.
6. View, and photograph.
Construct a restriction map of the plasmid. The data from the single digests indicates the number of cleavage sites
for each enzyme. Compare each single enzyme digest with the double digests using the same enzyme.
You should refer to the plasmid map and list of restriction enzymes recognition sites in the appendix for information
regarding the number and location of sites for the three restriction enzymes used above. This should allow you to
determine how successful you were in performing the digest reaction.
5B. Transformation
DNA molecules can be introduced into E. coli cells by three methods. Transduction is the introduction of genetic
information into a recipient cell by a bacteriophage. Conjugation involves the transfer of plasmid DNA from one cell
to another through cell-cell contact. Transformation results from a cell's uptake of purified DNA and is the method
with which we are concerned in this course. Some bacteria naturally take up exogenous DNA at certain stages of
growth; the ability to do so is called competence. E. coli, however, is not normally competent at any growth stage, but
competence can be induced by treating the cells with calcium chloride before adding the DNA. While the exact
Pg| 16
mechanism is not well understood it is thought the calcium ions complex with phosphates in the cell membrane and
destabilizes the membrane, causing pores to form. When DNA is added, calcium phosphate-DNA complexes cling to
the surface of the cell. Heat shocking constitutes a series of temperature changes (from ice to 42°C, then back to
ice). This is thought to create a current which draws the DNA into the cell. In this exercise, competent E. coli cells will
be transformed with plasmid DNA containing an antibiotic (Kanamycin or Ampicillin) resistance gene. We will
transform bacteria with the plasmid pAR 1.5. It was made by inserting a gene of interest (the PAR gene from the
microorganism Trypanosoma cruzi) into the plasmid pBK-CMV. Successful transformation will be detected by the
appearance of isolated colonies on antibiotic containing plates.
Protocol - Transformation
Use aseptic technique for all procedures!!
1. You will be provided a culture of E. coli DH5 in log phase of growth. Pour culture from the flask directly into
a large Sorvall tube up to the line visible about 3/4 inch from the top of the tube. Centrifuge at 5,000 rpm for
5 min. at 4°C. Discard the supernatant. (Note: save the remainder of the culture left in the flask-keep on ice)
2. Resuspend the pellet in 20ml of 50mM sterile CaCl2 by using a sterile pipette and working the liquid up and
down until the pellet is in suspension.
IMPORTANT: Look at the bottom of tube and make sure the pellet is completely resuspended.
3. Place on ice for 20-25 minutes.
4. Centrifuge at 5,000 rpm for 5 minutes at 4°C. Discard the supernatant.
5. Gently resuspend the pellet in 0.5ml of cold, sterile 50mM CaCl2. Again, make sure the pellet is completely
resuspended.
6. Obtain a vial of DNA (10µl of pAR 1.5 at 0.005µg/µl in 200mM CaCl2). Add 0.15ml of the competent cell
suspension from step 5 to the vial of DNA. Gently mix the calcium shocked cells.
7. In a separate tube, mix 10uL of sterile water and 0.15mL of the competent cell suspension from step 5.
This is your TRANSFORMATION CONTROL.
8. Gently mix by hand and place on ice for 10 minutes.
9. Transfer the vial to a 42° C water bath for 2 minutes.
10. Take a vial containing 2 ml of LB broth and pour all of it into the original vial containing your plasmid and
competent cells. Gently shake this culture for about an hour at 37°C in a shaking water bath.
11. Label the bottom of 4 petri dishes (2 containing plain LB and 2 containing LB + Ampicillin) with your
name and date. Label one of each (i.e. one plain, one w/ Amp.) with a "+". The remaining two plates should
be labeled with a "-".
12. After 1 hour, take two 0.1ml aliquots from the vial with competent cells + pAR 1.5 and place one aliquot in
the center of each of the first pair of plates (the ones labeled "+").
13. Take two 0.1 ml aliquots from the TRANSFORMATION CONTROL and place one aliquot in the center of
each of the second set of plates (the ones labeled "-"). Your instructor will demonstrate how to spread the
drops across the surface of the petri dish with the sterile spreader provided.
14. Place all 4 plates in the 37°C incubator.
Pg| 17
You will observe and analyze your plates at our next meeting.
Why are 4 plates used?
What results do you expect for each plate?
What do these results tell us?
The efficiency of transformation is the number of bacterial cells that are transformed per microgram of
plasmid DNA used. Rates usually range from 105 to 109. Calculate the efficiency of transformation for your
experiment. And remember to correct for the actual fraction of the cells (and hence, DNA!) plated.
Pg| 18
Lab 6
6A. Isolation of pAR 1.5 plasmid from successfully transformed cells
Your task is to isolate pAR1.5 DNA in order to analyze this plasmid by restriction digestion and to compare it to the
baseline pBK-CMV plasmid.
In the last lab, you transformed the pAR 1.5 plasmid into bacterial cells. We have generated cultures from
successfully transformed cells, and your goal in this part of the lab is to purify the pAR 1.5 plasmid from these cells.
There are several procedures available which allow isolation of plasmid DNA from bacterial cells. The one you will
use in this lab is based on one of the most popular methods, but adds a new way of actually purifying the plasmid
DNA. It is sold as a package under the name Qiagen System. Once the bacterial cells have been lysed, the cellular
debris (including bacterial DNA) is removed by centrifugation. The supernatant, containing the plasmid, is passed
over a minicolumn of DNA purification resin (it's a trade secret, but probably some kind of glass particles which easily
bind DNA). After the minicolumn is washed, the plasmid DNA is eluted with another buffer. The purified plasmid DNA
is now ready for RE digestion.
Protocol – Qiagen plasmid miniprep
Reagents:
Microcentrifuge tubes
Qiaprep spin column
Buffer P1: Resuspension buffer + RNase
Buffer P2: Lysis buffer
Buffer N3: Neutralization buffer
Buffer PB: Binding buffer
Buffer PE: Wash buffer
Buffer EB: Elution buffer
Procedure:
1. With a P-1000 micropipettor, aliquot 1.5ml of your culture into a microcentrifuge tube. Using a tabletop
centrifuge, spin cells for 1 minute at @ 13,000 rpm. With a pipette tip of a micropipettor, discard the
supernatant, try not to disturb the pellet).
2. Repeat step #1 twice (step #1 should be done a total of 3 times).
3. Resuspend pelleted bacterial cells in 250µl of Buffer P1 with a micropipettor tip or Pasteur pipette. Make
sure that there are no clumps remaining and transfer to a new microcentrifuge tube
4. Add 250µl of Buffer P2 to the microfuge tube and gently invert 4-6 times to mix. If necessary, continue
inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to
proceed more than 5 minutes.
5. To the microcentrifuge tube, add 350µl of Buffer N3 and invert the tube immediately (to avoid localized
precipitation) but gently 4-6 times. Mix solution gently but thoroughly. The solution should become cloudy.
6. Centrifuge for 10 minutes at maximum speed. A compact white pellet will form.
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7. With a micropipettor, carefully apply all of the supernatant to a Qiaprep spin column without disturbing the
pellet.
8. Centrifuge this for 30-60 seconds. Discard the flow-through.
9. Wash the Qiaprep spin column by adding 0.5ml of Buffer PB and centrifuge for 30-60 seconds. Discard
the flow-through.
10. Wash the Qiaprep spin column by adding 0.75ml of Buffer PE and centrifuge for 30-60 seconds.
11. Discard the flow-through and centrifuge for an additional minute to remove residual wash buffer. Residual
ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
12. Place the Qiaprep column in a clean 1.5ml microcentrifuge tube. To elute the DNA, add 60µl of Buffer EB
to the column and allow to stand for 1 minute.
13. Centrifuge for 1 minute.
14. Remove the column and save the flow-through.
15. You will use this DNA next week for your digests.
6B. Restriction Mapping of Plasmid DNA
Isolation of a plasmid from a transformed bacterium does not guarantee that the gene of interest has been cloned.
The next step in characterizing the recombinant is to create a restriction map. Restriction maps are constructed by
cutting DNA with one or more restriction enzymes to generate a series of fragments. The fragments are separated on
an agarose gel and the lengths determined by comparison to known molecular size standards. The fragment can
then be pieced together to construct a map. You did some of this earlier in the quarter, working with the pBK-CMV
plasmid (at that time it did not contain an inserted fragment of DNA). The plasmid, pAR1.5, should be a recombinant
pBK-CMV plasmid with a T. cruzi DNA insert at the polylinker site. This insert is the gene sequence for PAR2. The
main reason to do the digest is to obtain general information about pAR1.5. In addition, we might also glean
information about the size of the insert, and how to excise out the PAR2 gene from the plasmid.
When an insert is cloned into a vector, there is a 50% probability of the insert winding up in the proper orientation
with the promoter before the beginning of the gene sequence. There is also only a 1/3 chance of getting the correct
reading frame. This means an overall 1/6 chance of getting the correct protein. Directional cloning eliminates the
possibility of the insert winding up in the wrong orientation by using two different restriction enzymes to do the
cloning. When pAR 1.5 was created, the T. cruzi DNA was inserted by directional cloning. After the digests are
completed, you will analyze the fragments produced by agarose gel electrophoresis, determine the size of the
fragments, and construct a map of the recombinant plasmid.
In this lab, we will use the same three restriction enzymes that we used in the last lab: EcoRI, PstI, and SacI. We will
then compare the RE map generated in this lab with the RE map generated in the last lab to derive information about
the pAR 1.5 and its insert.
Pg| 20
Protocol – RE digestion of plasmid
1. Label 7 tubes – "S","E","P", "-", "S+E", "S+P", "E+P".
2. Use the tubes as a checklist for addition of reagents.
Tube
DNA
S
E
P
-S+P
S+E
E+P
5 µL
5 µL
5 µL
5 µL
5 µL
5 µL
5 µL
10X Buffer
1 µL A
1 µL H
1 µL H
1 µL H
1 µL A
1 µL A
1 µL H
SacI
EcoRI
1 µL
---1 µL
1 µL
--
-1 µL
---1 µL
1 µL
PstI
--1 µL
-1 µL
-1 µL
Water
3 µL
3 µL
3 µL
4 µL
2 µL
2 µL
2 µL
3. Use fresh tips for each new reagent. Add the enzyme last.
4. Mix and pool. Place the tubes in 37°C water bath for 60min.
5. Add 1µl load dye to each reaction. Load onto a 1% gel, run at 100V for 60 or more minutes. Also load
markers.
6. View and photograph.
Construct a restriction map of the plasmid pAR 1.5. The data from the single digests indicates the number of
cleavage sites for each enzyme. Compare each single enzyme digest with the double digests using the same
enzyme. How does this digest/map compare with the data from Lab 5, where you digested pBK-CMV alone?
Pg| 21
Lab 7
Micro RNAs (miRNAs) are a class of small (20-22
nucleotide) RNA molecules that were discovered
only recently (in 1993), but have since been shown
to be key regulators of gene expression with roles in
development, aging, cancer, and many other
diseases. It is now believed that as much as 4% of
the human genome could code for miRNAs!
miRNAs result from the processing of longer RNA
precursors that are cleaved by specific proteins into
the mature mRNAs. Figure 7-1 outlines the general
scheme for the generation of mature, active
miRNAs from their precursors. How the production
of miRNAs is controlled, and how specific miRNAs
are produced in response to specific signals is an
area of ongoing research.
Once produced, miRNAs can bind to
complementary sequences on their target mRNAs.
Thus, any mRNA (and be very careful with the use
of mRNA and miRNA – that one little “i” makes a
Figure 7-1: Generation of miRNAs and their effects on gene expression.
Taken from: http://pgfe.umassmed.edu/ou/wp-content/uploads/2012/09/image003.png
pretty big difference!) that contains a
complementary sequence to a particular miRNA is a
(potential) target for that miRNA (Figure 7-2). A miRNA works by recruiting a protein complex called the RISC (RNA
Induced Silencing Complex) to the miRNA targets. The RISC decreases expression of the target gene by a variety of
different mechanisms, the most important of which are translational repression and mRNA degradation (Figure 7-1).
It may therefore seem relatively straightforward to identify the targets of a given miRNA. All we would need to do is
search the genome of an organism for sequences that are complementary to our miRNA of interest. All the genes
that contain such complementary sequences would be targets of our miRNA. However, things are significantly more
complicated than this. Firstly, complete complementarity is not an obligate requirement for a miRNA to target a gene.
Even a partial match might be sufficient, and we do not understand the complete set of rules that govern target
choice for miRNAs. Secondly, genes that contain sequences that are highly complementary (though not completely
complementary) to a miRNA might not be targets for that miRNA (Figure 7-2). Thus, the identification of targets for a
specific miRNA is not a trivial task.
But why is it important to identify miRNA targets in the first place?
The answer lies in the importance of miRNAs to health and disease (for a listing of miRNAs associated with different
diseases, see http://www.mir2disease.org/). Since a single miRNA can regulate multiple genes, it is important to
know what the targets of a miRNA are, in order to understand its effects on the organism. For example, we can now
Pg| 22
convert skin cells into pluripotent stem cells simply
by treating the skin cells with the appropriate
combination of miRNAs. Or, if a certain disease is
caused by abnormal expression of a gene and we
know what miRNA targets this gene, then we could
inject this miRNA, causing the abnormally
expressed gene to be shut off, and in theory, cure
the disease. Alternatively, a disease could be
Figure 7-2: A single miRNA can have many targets. For simplicity, the
caused by misregulation of gene expression
miRNA is shown as a 8-mer (although miRNAs are usually 20-22nt in
because a particular miRNA is not being correctly
length). A given miRNA can target many genes (shown here as different
expressed. In these cases, the therapeutic strategy colored lines) as long as they contain a sequence that is complementary to
would be to change expression of the miRNA itself, the miRNA sequence. However, even partial complementarity (Genes D and
E) might be sufficient for a miRNA to target a particular gene. Other genes
thereby correcting the imbalance in gene
with partial complementarity (Gene F) are not targets of the miRNA.
expression, curing the disease. And in fact, miRNA
therapies are currently being investigated for a number of different diseases in labs all over the world (want to know
more? Start here: http://circres.ahajournals.org/content/110/3/496.full). In this lab, we will be attempting to identify the
targets of a miRNA called hsa-miR-23b-3p which will also be referred to in this manual simply as miR-23 (as a side
note on miRNA nomenclature, read this: http://www.mirbase.org/blog/2011/04/whats-in-a-name/). miR-23 is thought
to play some role in the development of different kinds of cancers, and overexpression of miR-23 might be able to
inhibit growth of tumors. Understanding how miR-23 affects the development and progression of cancer, therefore,
will help us develop new therapeutic strategies to deal with cancers that are affected by miR-23. The first step in this
process is to figure out what genes are regulated by miR-23.
As you go through this lab exercise, remember: This module is real, authentic research. No one knows all the targets
of miR-23, and someone in this class might be the first to discover a novel target of miR-23! How exciting is that?!!!
In order to find targets of miR-23, we will need to do the following:
1. First, pick a candidate gene that you think might be targeted by miR-23.
2. Design PCR primers specific to your candidate gene.
3. Extract total RNA from cells that are overexpressing miR-23 or a control miRNA.
4. Make cDNA from the RNA.
5. Measure the relative amount of your candidate gene’s mRNA using qPCR to determine if it is a target of
miR-23.
Pg| 23
7A: Picking a candidate gene
The first step in this experiment will involve picking a candidate gene. You will first use http://www.targetscan.org/
and http://pictar.mdc-berlin.de/cgi-bin/PicTar_vertebrate.cgi to identify likely targets of miR-23. These sites use
different bioinformatics algorithms to predict candidates that might be regulated by miR-23. When you use these
sites, remember to search specifically for hsa-miR-23. Otherwise, you might end up with irrelevant results! Play
around with these websites and explore the links. What information does each link give you? How might these
different bits of information be useful to you? Exploring the websites and the links is going to be important later
on, so don’t skip this step!
Select the species here
Maybe use this?
Or this?
Figure 7-3: Using TargetScan to find predicted targets of miR-23
Leave these defaults
Select miR
Figure 7-4: Using PicTar to find predicted targets of miR-23
Pg| 24
In using these resources to identify likely candidates, ponder the following questions:
 Why do the different websites return different results?
 Is one site better at predicting results than the other? How might you test if one site is better than the other?
 If a gene is predicted to be a target by both sites, is it a stronger candidate? How might your test this as a
general prediction?
As a researcher in the field of miRNAs, these are all questions that you would grapple with in the course of your
experiments, and to which, very often, there are no clear-cut answers. So, welcome to the world of real research!
Each group will pick one candidate gene to test. Some of the things to keep in mind when you are picking your
candidates are:
 Strength of prediction (does this even matter?!)
 What is known about the roles of miR-23 in cells?
 What is known about the roles of your candidate gene in cells?
 Is there a plausible connection between the roles of miR-23 and your candidate gene in cells? (Is this
important?!)
Keep in mind that the answers to these questions will be important to highlight in your lab report, when you describe
why you decided to test a particular candidate gene. Some resources that will help you answer these questions are:
 Pubmed
 Google Scholar
 Google Search
 Bing Search
Once you have picked a candidate gene, go ahead and claim it in the Google Docs spreadsheet (information on this
will be given to you in class/lab).
You cannot use a candidate gene that has already been claimed by another group. If all entries in the spreadsheet
are not filled in, then the gene has not been claimed! So make sure that you fill in all the columns to successfully
claim your candidate gene.
7B: Designing primers to test your candidate gene
Once you have claimed your candidate gene, the next step is to design primers that will be used to test the
expression of your gene of interest. The primers you design will be used to amplify your candidate gene by PCR
(recall what we have learned about PCR in the previous classes). The first thing you need to design primers is the
sequence of your candidate gene (will you use the genomic sequence of the gene, the mRNA sequence of the gene,
or the cDNA sequence of the gene? Why?). Obtain the correct sequence you are going to use from a link on the
candidate prediction website you used in part 7A (identifying your candidate gene). Check if there are alternate
versions of the gene you need to take into account (alternate splicing? RNA editing?). Then, figure out a good set of
primers to use. Ideally, your predicted PCR product should be around 150-250 bp in length.
Pg| 25
The parameters for good primers are (roughly in order of importance):
 The primers are SPECIFIC to your sequence (they don’t amplify anything else!)
 The primer pair (Forward + Reverse) shows no self-complementarity (why is self-complementarity bad?)
 The primer does not form step-loop structures
 The Tm of each primer is 55°-65°C
 Length is 18-30nt
 Usually ends in a C/G (called a CG-Clamp – why might this be good?)
 There are no large differences in the Tm between the forward and reverse primer
 There are no nucleotide “runs” (stretches of more than 3 of the same nucleotide) in the primers
It is certainly possible to manually stare at the candidate sequence and come up with primers that satisfy most of
these criteria. However, it is much easier and usually more accurate to use a computer to initially identify the best
candidates and then manually pick the best ones from this subset of primers. There are many, many, many different
algorithms and software that will pick primers for you, and most of them are very good. However, for this lab, we will
use www.bioinformatics.nl/primer3plus/ to design primers (Why you ask? –it's simple to use, it works pretty well,
and it's free!). Closely examine the output and pick a set of primers to use.
Click to search for primers
Enter sequence here
Figure 7-5: Using Primer3Plus to find primers for amplification of your candidate gene.
Double check the primers using:
Play with this to pick
the best settings;
what are you
designing the primers
for?
Figure 7-6: Optimizing settings for primer design in Primer3Plus using the “General Settings” tab
Pg| 26

http://www.basic.northwestern.edu/biotools/OligoCalc.html
Enter each primer, and use this program to calculate the primer parameters, and check the predicted selfcomplementarity (why might some of the parameters not match the Primer3 output?)
Enter single primer sequence here
a. Click here to get the thermodynamic
properties of the primer
b. Click here to check for primer
self-complementarity
Figure 7-7: Using OligoCalc to double check the primer characteristics
Pg| 27

http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Then, use the PrimerBLAST tool (BLAST is a way of checking for sequence similarity) to check if your primer pair is
specific (i.e., they don’t show amplification of other products).
Enter candidate gene
sequence here
Enter your primer
sequences here
Options. What do
you think they mean?
Figure 7-8: Checking for mispriming using PrimerBLAST
Using all these tools, you should have ensured that the primers satisfy the criteria listed above. (How will you decide
between pairs that appear very similar to each other?)
Once you’ve picked a set of primers, enter them in the Google Docs spreadsheet (information on this will be
provided in class/lab).
Make sure that all the columns of information are filled out correctly. Most importantly, make sure you have entered
the correct primer sequence (probably best to use copy-and-paste from the primer prediction website), and that it is
in the correct orientation. We will order the primers for you, and they will be ready to use in the next lab.
Pg| 28
Lab 8
8A: Isolation of total RNA from cells
Now that you have chosen your candidate gene and designed primers specific for it, the next step is to isolate RNA
from HeLa cells that have already been transfected with miR mimics for either miR-23 or a control “scramble”
sequence. Mimics are short double-stranded RNAs that are designed to mimic an endogenous miRNA, which
causes an up-regulation of the miRNA’s activity. The scramble mimic is designed specifically to not mimic any known
endogenous miRNA. In order to generate your samples, HeLa cells were transfected using DharmaFECT1
Transfection Reagent from Thermo Scientific. This transfection process (there are many others including chemicalbased, electroporation, optical with lasers, magnet assisted, and more) uses liposomes to introduce the miR-mimic
and scramble RNA into the cells by taking advantage of the negative charge of the RNA. (Why might one transfection
process be better than another?) The lipids in the transfection reagent form a vesicle around the RNA, which then
fuses to the phospholipid bilayer and releases the genetic material into the cells. At 48 hours post-transfection the
cells are passaged (cells are allowed to grow in fresh media), and then aliquoted at 5x105 cells per microfuge tube
and pelleted by centrifugation. The supernatant was discarded and the pellet was frozen at -80ºC.
On the day of the lab the cells will be lysed and the RNA will be isolated and purified using the GeneJET RNA
Purification Kit from Thermo Scientific. (Why do we need to purify the RNA?) The lysis buffer used in this kit contains
guanidine thiocyanate, which is a chaotropic salt that disrupts and denatures RNases. When mixed with ethanol, this
salt causes the RNA to bind to the silica membrane on the column used during washing. Impurities can then be
removed using wash buffers and centrifugation. What causes the RNA to remain on the column during the washing
steps? The ethanol and high ionic strength of the wash buffers!
In the final step, the RNA is eluted using nuclease-free water (low ionic strength) and collected in a clean
microcentrifuge tube. Once we have purified the RNA we will use it to make cDNA.
To purify your RNA from your cell pellets using the GeneJET RNA Purification Kit from Thermo Scientific you will
need six ingredients:
1. Frozen Cell Pellets in microcentrifuge tubes (1 for the miR-scramble and 1 for the miR-23 mimic)
2. Lysis buffer supplemented with β-mercaptoethanol (why do we add β--mercaptoethanol?)
3. Ethanol (what happens to the structure of RNA when ethanol is added?)
4. Wash Buffer 1 supplemented with 96-100% ethanol
5. Wash Buffer 2 supplemented with 96-100% ethanol
6. Water, nuclease-free (why does the water have to be nuclease-free?)
Pg| 29
Protocol – RNA isolation
NOTE: Each group will make RNA from ONE sample (either the miR-treated sample, or the control)
1. Resuspend cell pellet in 600µL of Lysis Buffer by pipetting. Vortex for 10 seconds.
2. Centrifuge for 5 min at 14000xg and transfer the supernatant to a new sterile RNase-free microcentrifuge
tube.
3. Add 360µL of (96-100%) ethanol to each tube and mix by gently pipetting.
4. Transfer 700 µL of each lysate to a Purification Column inserted in a collection tube.
5. Centrifuge for 1 min at 12000xg. Discard the flow-through in the collection tubes and place the purification
columns back into the collection tubes. Repeat steps 4 and 5 until all of the lysate has been purified (at this
point our RNA is attached to the silica membrane in the purification column).
6. Add 700 µL of Wash Buffer 1 to the columns.
7. Centrifuge for 1 minute at 12000xg. Discard the flow-through in the collection tubes and place the
purification columns back into the collection tubes.
8. Add 600 µL of Wash Buffer 2 to the columns.
9. Centrifuge for 1 minute at 12000xg. Discard the flow-through in the collection tubes and place the
purification columns back into the collection tubes.
10. Add 250 µL of Wash Buffer 2 to the columns.
11. Centrifuge for 2 minutes at 12000xg. Discard the flow-through in the collection tubes and place the
purification columns back into the collection tubes.
12. Re-centrifuge the columns for 1 minute at 14000xg. (Why do we do this step?)
13. Place the purification column into a sterile 1.5 mL RNase-free microcentrifuge tube. You can now discard
the collection tube, because our RNA is ready to be eluted from the membrane on the column.
14. Add 50 µL of nuclease-free water to the center of the silica membrane of the purification columns. This is
the elution step, so the water must be in contact with the membrane where the RNA is. While adding the
water, make sure that you DO NOT touch the membrane with your pipette tip.
15. Incubate at room temperature for 3 min.
16. Centrifuge for 1 minute at 12000xg to elute the RNA into the sterile 1.5 mL RNase-free microcentrifuge
tube. Retain the purification column in case an additional elution step is needed (it can be discarded at the
end of class).
17. Split the RNA into two aliquots, and immediately freeze one aliquot at -20°C.
18. Congratulations! You now have purified RNA. Proceed to performing reverse transcription to create cDNA.
Pg| 30
8B: Making cDNA from total RNA
Once the RNA is isolated, it should represent a snapshot of gene expression (mature mRNA) in cells treated with
either miR-scramble control or miR-23. Our overall goal is to use the primers designed in lab 7 to detect relative
expression of your candidate gene in miR-23 treated cells compared to miR-scramble control treated cells. In this
section you will take the isolated RNA and convert it into complementary DNA (cDNA). Why can’t we just use the
isolated RNA and “count” the copies of our candidate gene? There are two reasons. First, single stranded RNA is
highly unstable meaning that it is sensitive to degradation by RNases (enzymes that degrade single stranded RNA).
Secondly, the method used to detect relative expression of your candidate gene is based on a fluorescent dye
inserting itself (intercalating) between double stranded DNA.
Beginning with single stranded RNA, we will take advantage of reverse transcriptase, an enzyme isolated from a
virus that uses an RNA template to generate a complementary DNA strand. All DNA polymerases are primer
dependent and so we use both oligo (dT)18 which is a short sequence of 18 deoxy-thymine nucleotides (why would
we use this and where on the mRNA would it bind to?) as well as random hexamers which are a mixture of singlestranded random hexanucleotides (where on the mRNA would these bind to? Why do we need a mixture of
hexamers?). In order to synthesize cDNA, the enzyme needs the raw material or building blocks for the new cDNA
strand and so we also add a mixture of the fours dNTPs.
RNases exist everywhere and are a common defense in the human body to destroy free-floating single stranded
RNA that is generally viral in origin. One precaution when working with RNA, and when performing the cDNA
synthesis is to not talk, breathe, sneeze, or cough over your tubes, otherwise RNases could make it into your
tube and destroy your RNA!
In order to generate cDNA you will use the Maxima First Strand cDNA Synthesis Kit from Thermo Scientific. To setup
the reaction you will need four ingredients:
1. 5X reaction buffer. This contains your reaction buffer (salts), dNTPs, oligo dT and random hexamer primers.
2. Maxima enzyme mix. This contains the reverse transcriptase as well as RNase inhibitor.
3. Water, nuclease-free (why does the water have to be nuclease free?)
4. Template RNA. Single stranded RNA isolated in 8A.
Note: It is very important to keep anything with the Maxima enzyme mix on ice until the reaction is ready to
place into the thermocycler.
Pg| 31
Protocol – cDNA synthesis
1. NOTE: Get the RNA sample for the other sample from another group (so, if you made RNA from the
miR-treated sample, get the RNA for the Control samples from another group). This way, each group will
have a complete set of samples to compare.
2. Add the reagents shown in the table into sterile, RNase-free PCR tubes on ice in the indicated order (from
left to right). Why do we include the tubes without the reverse transcriptase enzyme mix added (-RT)?
Tube
Sample
template
5X Rxn
buffer
Maxima
enzyme mix
Template
RNA
Water,
nuclease-free
Total volume
C+RT
Ctl
4 µl
2 µl
1 µl
13 µl
20 µl
C-RT
Ctl
4 µl
0 µl
1 µl
15 µl
20 µl
M+RT
miR
4 µl
2 µl
1 µl
13 µl
20 µl
M-RT
miR
4 µl
0 µl
1 µl
15 µl
20 µl
3. Cap tubes and flick gently to mix. Briefly centrifuge to collect reaction to the bottom.
4. Place tubes in thermocycler using the following protocol:
10 min at 25˚C. 15 min at 50˚C. 5 min at 85˚C. Hold at 4°C.
Now you have cDNA! Proceed to the qPCR.
8C: Quantifying gene expression using qPCR
Gene “Blue” starts with only one copy
Gene “Green” starts with three copies
Figure 8-1: The initial number of PCR products is very sensitive to the amount of starting template. The “Blue” gene has only one cDNA
molecule, and at the end of 3 cycles of qPCR results in 4 ds products. However, the “Green” gene starts off with 3 copies, and at the
end of 3 cycles of qPCR results in 12 ds products. Thus, measuring the amount of product in the early rounds of PCR gives us a very
accurate and sensitive estimate of the starting template amount.
Pg| 32
You have finally synthesized your cDNA from the RNA isolation step - but now what? Now it’s time to start the actual
experiment of quantifying the candidate gene expression in the lysed cells treated with either miR-scramble control or
miR-23. Compared to RNA, the cDNA synthesized is quite stable, as it exists as tightly paired double stranded
structure. Though relatively stable at 4°C, properly stored cDNA has been found to be useable even after 16 years!
You will quantify gene expression using the primers you designed in Lab 7 to specifically amplify your candidate gene
by PCR. You will employ a variation of PCR called quantitative PCR (qPCR) to estimate the relative expression
level of your candidate gene. qPCR couples DNA amplification with detection in real time (will you need to run a gel
at the end of qPCR?) to give a very accurate estimate of the amount of target sequence present in the PCR reaction.
Remember that in the initial stages, the rate limiting step in PCR is the amount of template DNA (Why?), so the
amount of product formed will be directly proportional to the amount of template DNA present. Thus, by measuring
the amount of product in the early cycles of PCR, we can get a very accurate and sensitive estimate of the amount of
template present in the reaction (Figure 8-1). If your candidate gene was a target of miR-23, you would then expect
to see a difference in the amount of the starting cDNA template (and hence, the original mRNA) between the miR-23treated cells, and the controls (what difference do you expect to see?).
We will be using fluorescent dyes in our qPCR to label the PCR products produced and quantify the relative amounts
of amplified target gene DNA. Specifically, we will use “SYBR Green”, which binds to all double-stranded DNA and
detection of your target gene is monitored at each PCR cycle by increases in fluorescence (see Figure 8-2).
Before one starts the qPCR with their cDNA, the issue of normalization needs to be addressed. Imagine a scenario
where you find that your miR-treated cells have 50% of you candidate mRNA when compared to controls. However,
you also started off with 50% less total RNA in your miR-treated cells when
compared to controls. Now, can you make any conclusions about your
candidate gene? Therefore, when doing qPCR, one needs to be assured
that the total amount of input RNA is equal across all samples (or that we
can normalize our final values based on the amount of total starting RNA).
This doesn’t mean that we are looking to make sure that the starting RNA of
your actual target gene is the same (then there would be no difference in
gene expression!), rather we are looking to make sure the total cellular
amounts of RNA from your cells is comparably equal. To do this, a
housekeeping gene control is introduced across all the samples to be run,
and we pick a gene that is known to NOT be targeted by our miR (why is it
important that the gene not be target?). A housekeeping gene is defined as
any reference gene found in your cell type that will not fluctuate in
expression levels when subject to the same treatments or conditions that
one has performed on the cells. For our experiment, we will be using
Beta-2 microglobulin, a MHC class I molecule present in all nucleated cells.
Further, we will need to include a positive control, to make sure that our
experimental conditions are working. What do you think is a good positive
control for our experiment?
Figure 8-2: Principle for SybrGreen qPCR.
Pg| 33
To set up the qPCR, we will use the Thermo Scientific Luminaris Color Hi ROX qPCR Master Mix from Thermo
Scientific. Here is what is needed to setup the reaction:
1. 2X qPCR master mix (contains your Hot Start Taq DNA polymerase, dNTPs, and optimized PCR buffer)
2. Water, nuclease-free
3. 40X Sample Buffer
4. Template cDNA obtained from 8B
5. Forward primers
6. Reverse primers
Note: Keep everything on ice until the reaction is ready to place into the thermocycler.
Protocol - qPCR
1. NOTE: If you have not filled in the information in the spreadsheet correctly, we cannot track your samples,
and you will not get any results back.
2.
Prepare your template DNA for each sample (+RT or –RT) using the table below.
Tube
Sample
Water
DNA
40X Yellow Sample Buffer
1
2
3
4
C+RT (Ctl)
C-RT (Ctl)
M+RT (miR)
M-RT (miR)
16µL
16µL
16µL
16µL
4µL
4µL
4µL
4µL
5µL
5µL
5µL
5µL
This diluted, yellow sample is referred to as the “Final sample.”
3. Prepare your primers. The primers you designed have been synthesized, but are in the form of a salt. Check
the amount of the forward and reverse primer that you have (the amount is printed on the label on the
primer tube), and use this to add enough water to get a final primer concentration of 100µM. Show your
calculations to your TA before adding the water.
4. Use your diluted primers to make 10µL of a primer mix containing 10µM each of the forward and reverse
primer. Show your calculations to your TA before making your primer mix.
5. Similarly, make 10µL of a primer mix containing 5µM each of the forward and reverse primers for the B2M
primers. Assume that the primers were initially at a concentration of 10µM each. Show your calculations to
your TA before making your primer mix.
6. Finally, make 10µL of a primer mix containing 5µM each of the forward and reverse primers for the MAP3K1
primers. Assume that the primers were initially at a concentration of 10µM each. Show your calculations to
your TA before making your primer mix.
Pg| 34
7. Add the reagents shown in the table below into a sterile, 8-strip PCR tube on ice in the indicated order (from
left to right). Keep track of what sample is in which tube by correlating the tube number with the sample in it.
1
Primers
(F+R mix)
2X Master Final
Water,
mix
Sample nuclease-free
Tube
Template
1
+RT (Ctl)
B2M – 2.5 µL
10 µl
2.5 µL
5.0 µL
2
+RT (Ctl)
MAP3K1 - 2.5 µL
10 µl
2.5 µL
5.0 µL
3
+RT (Ctl)
Candidate gene - 1.5 µL
10 µl
2.5 µL
6.0 µL
4
-RT (Ctl)
B2M - 2.5 µL
10 µl
2.5 µL
5.0 µL
5
+RT (miR)
B2M - 2.5 µL
10 µl
2.5 µL
5.0 µL
6
+RT (miR)
MAP3K1 - 2.5 µL
10 µl
2.5 µL
5.0 µL
7
+RT (miR)
Candidate gene - 1.5 µL
10 µl
2.5 µL
6.0 µL
8
-RT (miR)
B2M - 2.5 µL
10 µl
2.5 µL
5.0 µL
2
B2M = Beta-2-microglobulin, the endogenous control; MAP3K1 = Positive control
IMPORTANT: Remember to label your tubes with your unique Group number or we will not be able
to identify your samples (See example image below)!!!
IMPORTANT: Do NOT label the caps of the tubes or the qPCR machine CANNOT read the
fluorescence in your samples!
Figure: Example of qPCR tube labeling.
DO NOT label the lids. You need to label the
tubes with a tube number (and keep track of
what sample is in which tube), and your
unique group number.
REALLY IMPORTANT: Do NOT label the caps of the tubes!
8. Cap tubes and flick gently to mix. Briefly centrifuge to collect reaction to the bottom. Make sure all the tubes
have a green solution, and that all tubes contain the same volume of contents!
9.
Keep your tubes on ice. Your TA will take your tubes to the qPCR machine (which is essentially a
thermocycler/PCR machine with some fancy optics to measure fluorescence in real time) and run your
samples using the following program:
10 min at 95˚C
15 sec at 95˚C, 30 sec at 60˚C, and 30 sec at 70˚C - for 40 cycles
Hold at 4°C.
10. We will send you the results of the qPCR. And you get to analyze them!
IMPORTANT: Remember to label your tubes with your unique Group number!!! See figure above.
For the next lab, you will repeat the RT-PCR and qPCR, but get the RNA
samples from a group whose RNA was not contaminated.
Pg| 35
Using your qPCR results (CT
method5)
You must calculate the Delta Delta CT for your target gene and the Positive control using the following information:
CT = CT(control) - CT(miR)
Where:
CT(control) = CT(target-control) - CT(endogenous control-control)
CT(miR) = CT(target-miR) - CT(endogenous control-miR)
You can then calculate the ratio of expression as 2CT
Remember that higher expression means that your threshold cycle will be lower! Also pay attention to your –RT
readings. What does it mean if they are high? What does it mean if they are low?
Worked example:
miR
Sample
Endogenous
Positive
Target
Endogenous
Control
Sample
CT
CT
14.7492
Endogenous
15.5057
28.1164
Positive
26.7152
28.8212
Target
25.4664
Endogenous
Undetermined
Undetermined
(Note: Undetermined = no amplification)
miR
CT(target)
= 28.8212 – 14.7492 = 14.072
CT(positive) = 28.1164 – 14.7492 = 13.3672
Control
CT(target)
= 25.4664 – 15.5057 = 9.9607
CT(positivel)
= 26.7152 – 15.5057 = 11.2095
CT(target) = 9.9607 – 14.072
= -4.1113
Ratio of expression = 2-4.1113 = 0.056
CT(Positive)= 11.2095 – 13.3672
= -2.1577
Ratio of expression = 2-2.1577 = 0.224
Therefore Target IS decreased by miR overexpression (positive control worked, shows decreased
expression).
5
Adapted from http://blog.mcbryan.co.uk/2013/06/qpcr-normalisation.html
Pg| 36
Supporting Information
Pg| 37
Example Restriction Enzyme Information
Pg| 38
Example RE information from NEB website:
http://www.neb.com/nebecomm/products/category1.asp#2
HindIII has a High Fidelity (HF) Version available
HindIII
Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the HindIII gene from Haemophilus influenzae Rd (ATCC 51907).
Reagents Supplied:
NEBuffer 2
Enzyme Properties
Activity in NEBuffers:
NEBuffer 1: 50%
NEBuffer 2: 100%
NEBuffer 3: 10%
NEBuffer 4: 50%
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme
to achieve complete digestion.
Methylation Sensitivity:
dam methylation: Not sensitive
dcm methylation: Not sensitive
CpG methylation: Not sensitive
More information about: Methylation Sensitivity
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
(+ + +) Suitable for an extended or overnight digestion. Enzyme is active > 8 hours.
More information about: Extended Digests with Restriction Enzymes
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 2
Incubate at 37°C.
1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl
2
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total
reaction volume of 50 µl.
Concentration:
20,000 units/ml and 100,000 units/ml
Unit Assay Substrate:
λ DNA
Pg| 39
Storage Conditions:
10 mM Tris-HCl
250 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
500 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent B
Notes
General notes:
1. Not sensitive to dam, dcm or mammalian CpG methylation.
2. Conditions of high enzyme concentration, glycerol concentration > 5% or pH > 8.0 may result in star
activity.
FAQs
1. Will BSA affect HindIII activity?
2. What is Star Activity and how can it be avoided?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Ligation and Re-cutting:
After a 100-fold overdigestion with HindIII, > 95% of the DNA fragments can be ligated with T4 DNA
Ligase (at a 5' termini concentration of 1-2 μM) at 16ºC. Of these ligated fragments, > 95% can be recut
with HindIII.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA and 400 units of HindIII incubated for 16 hours at 37ºC resulted in
a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. Note
that the high enzyme concentration described in this assay may result in star activity.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 2,000 units of HindIII with 1 μg of a mixture of single and double3
5
stranded [ H] E. coli DNA (10 cpm/μg) for 4 hours at 37ºC released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 40 units of HindIII with 1 μg of ΦX174 RF I DNA for 4 hours at
37ºC resulted in < 10% conversion to RFII as determined by agarose gel electrophoresis.
Blue/White Screening Assay:
An appropriate vector is digested at a unique site within the lacZα gene with a 10-fold excess of enzyme.
The vector DNA is then ligated, transformed, and plated onto Xgal/IPTG/Amp plates. Successful expression
of β-galactosidase is a function of how intact its gene remains after cloning, an intact gene gives rise to a
blue colony, removal of even a single base gives rise to a white colony. Enzyme preparations must
produce fewer than 3% white colonies to be Blue/White certified.
Pg| 40
HindIII crystals (Ira Schildkraut and Lydia Dorner, New England Biolabs)
Reagents Sold Separately
NEBuffer 2
Pg| 41
DNA Ladders used in experiments
Pg| 42
5µl ladder
10µl ladder
These two ladders might be interchanged!
Look at the banding patterns of your
ladders to decide which of these
correspond to your 5µL and 10µL ladders!
Pg| 43
HinDIII digested Lambda DNA ladder
The DNA ladder generated by digestion of
Lambda DNA with HinDIII is shown. NOTE
that this is after heating the digestion and
then running on a gel. Since we did not heat
the digest, you may not see all the bands
shown here. This is an image taken from the
GenScript* website.
* http://www.genscript.com/molecule/MM1212-Lambda_DNA_HindIII_Marker_ready_to_use_sup_TM_sup_.html
Pg| 44
pBK-CMV information
Pg| 45
Pg| 46
Name RE Site
Cut
#Sites Sites
Name
RE Site
#Sites Cut Sites
AccI
GTMKAC
1
1105
StuI
AGGCCT
1
4050
ApaI
GGGCCC
1
1046
TspGWI
ACGGA
1
2998
ApaLI
GTGCAC
1
2263
Tth111I
GACNNNGTC
1
3752
AsuII
TTCGAA
1
3187
VspI
ATTAAT
1
1886
BalI
TGGCCA
1
3789
XbaI
TCTAGA
1
1056
BamHI
GGATCC
1
1092
XhoI
CTCGAG
1
1068
BclI
TGATCA
1
692
AflIII
ACRYGT
2
463, 1949
BsePI
GCGCGC
1
1116
AjuI
GAANNNNNNNTTGG
2
718, 750
BseRI
GAGGAG
1
4055
AloI
GAACNNNNNNTCC
2
276, 308
BsrDI
GCAATG
1
3640
AlwNI
CAGNNNCTG
2
2365, 2773
BstXI
CCANNNNNNTGG
1
1028
AvaI
CYCGRG
2
1024, 1068
BtsI
GCAGTG
1
516
AvaII
GGWCC
2
2907, 3352
DraIII
CACNNNGTG
1
240
AvrII
CCTAGG
2
3030, 4045
Eco31I
GGTCTC
1
2878
BsaBI
GATNNNNATC
2
691, 4012
EcoRI
GAATTC
1
1083
BsaXI
ACNNNNNCTCC
2
276, 306
HindIII
AAGCTT
1
1074
BsmI
GAATGC
2
511, 604
HpaI
GTTAAC
1
590
ClaI
ATCGAT
2
1037, 4029
KpnI
GGTACC
1
1019
CspCI
CAANNNNNGTGG
2
1486, 1521
MfeI
CAATTG
1
599
DraII
RGGNCCY
2
720, 2770
MluI
ACGCGT
1
463
FalI
AAGNNNNNCTT
2
3536, 3568
NarI
GGCGCC
1
3869
HindII
GTYRAC
2
590, 1106
NdeI
CATATG
1
1659
PasI
CCCWGGG
2
2800, 2903
NheI
GCTAGC
1
1300
PsiI
TTATAA
2
365, 570
NotI
GCGGCCGC
1
1049
SapI
GCTCTTC
2
3314, 3524
OliI
CACNNNNGTG
1
1026
SexAI
ACCWGGT
2
1011, 4277
PfoI
TCCNGGA
1
3091
SspI
AATATT
2
445, 4410
PstI
CTGCAG
1
1114
BciVI
GTATCC
3
2152, 3653, 4443
PvuI
CGATCG
1
852
BsaAI
YACGTR
3
237, 1555, 3567
RsrII
CGGWCCG
1
3352
BspHI
TCATGA
3
695, 2669, 4441
SacI
GAGCTC
1
1126
DrdI
GACNNNNNNGTC
3
284, 2057, 3846
SacII
CCGCGG
1
1049
Eco57I
CTGAAG
3
2497, 3295, 3727
SalI
GTCGAC
1
1104
GsuI
CTGGAG
3
1110, 3033, 3090
ScaI
AGTACT
1
1064
MmeI
TCCRAC
3
262, 2164, 2348
SfiI
GGCCNNNNNGGCC
1
4099
MslI
CAYNNNNRTG
3
1026, 1530, 3434
SmaI
CCCGGG
1
1026
NaeI
GCCGGC
3
134, 3086, 3369
SnaBI
TACGTA
1
1555
NcoI
CCATGG
3
1531, 3435, 4138
SpeI
ACTAGT
1
1098
NmeAIII
GCCGAG
3
3648, 4084, 4119
Sse8387I
CCTGCAGG
1
1114
PvuII
CAGCTG
3
881, 3765, 4373
Pg| 47