PCR Optimization

Table of Contents
Fall 2012




Optimizing the Polymerase Chain Reaction

Introduction .....1
Review of Mathematics ........3
Solving Problems of Dilution and Concentration: Two Approaches ..4
Experiment Overview 7
Calculations
Part 1: Overview of PCR Reaction Components ....8
Part 2: Varying the Magnesium Chloride Concentration ..10
Part 3: Varying the Primer Concentration ..15
Laboratory Exercise.20
Important Laboratory Practices .20
Varying Magnesium Concentration ..21
Varying Primer Concentration ...24
Agarose Gel Electrophoresis .27
Electrophoresis of Amplified DNA .28
Staining and Photographing Agarose Gels .29
Interpretation of Results
Role of Magnesium 30
Role of Primers ...32
Acknowledgements .33



PCR Optimization
Student Guide
Fall 2012
1

Optimizing the Polymerase Chain Reaction


Frank H. Stephenson, Ph.D. Maria C. Abilock
Applied Biosystems BABEC


Introduction

The Polymerase Chain Reaction (PCR) is a powerful technique used for the amplification of a specific segment of
a nucleic acid. Starting with only a very small amount of material, a DNA segment can be multiplied by over a
million-fold. Because of this great sensitivity, PCR has found popularity in a wide range of applications. Molecular
biologists use PCR in gene cloning and DNA sequencing. Forensic scientists use PCR to connect blood, semen,
saliva, or tissue left at the scene of a crime to a suspect or victim. Clinical geneticists use PCR to determine
whether or not potential parents might carry a genetic disease that could be passed along to their children.

PCR is DNA synthesis in a test tube. To perform PCR amplification, you need to combine all the components
required for DNA replication. These are as follows:

Template DNA. The template carries the DNA segment or target you wish to amplify.
Primers. A primer is a short, single-stranded piece of DNA that anneals (attaches) to its complementary
sequence on the template. A pair of primers will bind to either side of the target DNA segment providing
initiation sites for DNA synthesis. The two primers used in a PCR are often designated by the terms forward
and reverse.
DNA Polymerase. This is the enzyme used to synthesize new strands of DNA. DNA polymerase adds
nucleotides onto the end of an annealed primer. The added bases are complementary to the template strand.
Magnesium. DNA polymerase requires magnesium for activity. Magnesium is usually supplied to a PCR
amplification in the form of magnesium chloride.
dNTPs. dNTP stands for deoxynucleoside triphosphate. These are the four nucleotides used by DNA
polymerase to extend an annealed primer.
Buffer. A buffer is a solution that resists change in pH. It is used to ensure that the DNA polymerase is in an
environment in which it will have maximum activity.

Although PCR is a powerful tool for the amplification of genetic sequences, it is not an exact science. Sometimes,
even though using an established PCR protocol that had been optimized and successful for the amplification of a
particular DNA segment, use of that same protocol on a different region can result in a less than desirable outcome.
Poor amplification, however, can result from one of several causes; the temperatures used for thermal cycling may
not be the best, the template may be of poor quality, or a reagent (or reagents) may be at suboptimal
concentration(s).

The optimal reagent concentrations for any specific target/primer set and the optimal thermal cycling parameters
that will give the highest yield, more often than not, are determined by trial and error. Taking particular care when
optimizing PCR can provide rewards in several ways. An optimized PCR will improve both product yield and
reproducibility between reactions, while reducing amplification of non-specific products. When electrophoresed on
an agarose gel, an optimized reaction will give a brighter product band with minimal background (Figure 1).

When developing a protocol for PCR amplification of a new target, it may be important to optimize all parameters
including reagent concentrations, cycling temperatures, and cycle number. For this experiment, thermal cycling
parameters have been optimized for you. Your task will focus on reagent optimization. Varying the magnesium or
primer concentration usually has the most profound affect on the quality of the reaction. Therefore, your class will
optimize these reagents in a PCR experiment. To do this, you will amplify a segment of the acetylcholinesterase
gene in cow DNA.


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Student Guide
Fall 2012
2




Figure 1. An optimized PCR amplification will produce a single, bright band on a gel (as seen in the left-most lane).
A reaction for which conditions have not been optimized will produce multiple bands as seen in the center lane. The
right-most lane contains size marker.

PCR Optimization
Student Guide
Fall 2012
3

Review of Mathematics

This laboratory exercise requires that you calculate amounts and concentrations of various reagents.
Concentration refers to the amount of a substance dissolved in a defined volume. Two teaspoons of sugar
dissolved in a cup of water is an example of a concentration. Two teaspoons of sugar in one cup of water is more
concentrated than one teaspoon.

If you make a reagent less concentrated by transferring a small amount into a new volume, you have made a
dilution. For example, you dilute liquid dishwashing soap by pouring a small amount into a sink full of water.

In PCR, the concentration of the individual reaction components may be expressed in different units. For example,
the amount of template DNA is calculated as ng/reaction, while the concentration of primer is expressed as
micromolar (!M). To perform the calculations in this protocol, you need to be comfortable using the different units
and converting between units.

When changing between units, you must use a conversion factor, or a numerical ratio equivalent to one. For
example, 100 pennies make up one dollar. The conversion factor is

100 pennies
1 dollar

How would you use this conversion factor to solve the problems below? Show your work.

1. How many pennies are represented by 15 dollars?

15 dollars x 100 pennies / 1 dollar = 1500 pennies

2. How would you determine the number of dollars equivalent to 800 pennies?

800 pennies x 1 dollar / 100 pennies = 8 dollars

Below is a chart showing conversion factors to help you change between units for volume measurements. Lets try
some examples. Show your work.

Unit Symbol Unit Equivalence Conversion Factors
milliliter mL 1,000 mL = 1 liter (L) 1,000 mL or 1 L
1 L 1000 mL
microliter !L 1,000,000 !L = 1 L 1,000 !L or 1 mL
1 mL 1000 !L
Table 1: Conversion Factors for Volume Measurements


3. How many !L are equivalent to 3.3 milliliters?

3.3 mL x 1000 !L / 1 mL = 3300 !L

4. Express 450 !L as milliliters.

450 !L x 1 mL / 1000 !L = 0.45 mL

When you need to convert between units for measurements of mass, Table 2 may be helpful.
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Student Guide
Fall 2012
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Unit Symbol Unit Equivalence Conversion Factors
milligram mg 1,000 mg = 1 gram (g) 1000 mg or 1 g
1 g 1000 mg
microgram !g 1,000,000 !g = 1 g 1000 !g or 1 mg
1 mg 1000 !g
nanogram ng 1,000,000,000 ng = 1 g 1000 ng or 1 !g
1 !g 1000 ng
Table 2: Conversion Factors for Mass Measurements

Use the conversion factors from the chart above to change between mass units. Show your work.

5. How many micrograms are equivalent to 0.25 g?

0.25 g x 1,000,000 !g / 1 g = 250,000 !g

6. Express 336 ng in terms of !g.

336 ng x 1 !g / 1000 ng = 0.336 !g


In your optimization experiment, you will be figuring out the concentrations of reagents needed for this particular
PCR protocol. To help you convert between units of concentration, you may need Table 3. In the next set of
practice problems, please make sure to show your work.

Unit Symbol Unit Equivalence Conversion Factors
millimolar mM 1,000 mM = 1 molar (M) 1000 mM or 1 M
1 M 1000 mM
micromolar !M 1,000,000 !M = 1 M 1000 !M or 1 mM
1 mM 1000!M
Table 3: Conversion Factors for Concentration Measurements


7. Convert 500 !M to the equivalent unit expressed in mM.

500 !M x 1 mM / 1000 !M = 0.5 mM

8. How would you express 0.04 mM as !M?

0.04 mM x 1000 !M / 1 mM = 40 !M
PCR Optimization
Student Guide
Fall 2012
5

Solving Problems of Dilution and Concentration: Two Approaches

Everyone has a slightly different manner in which they think about numbers and mathematical relationships. It is
one of the beauties of mathematics that any one of several different approaches can often be used to solve a
problem and there is not necessarily any one correct way to arrive at the solution.

Most of the problems you will encounter in preparing for this laboratory exercise involve calculating the dilution
required to bring a particular PCR reagent to a new, desired concentration within a reaction. For example, lets say
that you have a stock solution of 5 mM (millimolar) magnesium chloride (MgCl
2
) and need to add some volume of
that stock solution to a 200 !L reaction such that that reaction has a final concentration of 100 !M MgCl
2
. How
much of the stock solution should you add? The two most popular approaches for tackling this problem will be
presented here.

Approach I: (C
i
)(V
i
) = (C
f
)(V
f
)

The formula above states that the concentration of the reagent in the initial stock solution (C
i
) multiplied by a certain
volume (V
i
) should equal the reagents final, diluted concentration (C
f
) multiplied by the final volume (V
f
) of the
reaction containing the diluted reagent. For most of the problems in this protocol, you will be solving for V
i
. To solve
a problem using this equation, you must first identify each term. You must also make sure that all terms are in the
same units. If they are not, they must be converted prior to solving the equation.

For our example, we define each term as follows:

C
i
= 5 mM MgCl
2

V
i
= unknown
C
f
= 100 !M MgCl
2

V
f
= 200 !L

Notice that C
i
is in units of mM and C
f
is in units of !M. C
i
can be converted to units of !M as follows:




The problem can now be solved:

(5000 !M)(V
i
) = (100 !M)(200 !L)
V
i
= (100 !M)(200 !L) / 5000 !M = 4 !L

Therefore, you would add 4 !L of 5 mM MgCl
2
to a 200 !L reaction to give a final concentration of 100 !M MgCl
2
.

Approach II: Dimensional Analysis and Canceling Terms

As a variation of Approach I, an equation can be written such that all units of concentration on the left side of the
equation cancel accept for the one that represents that of the final concentration written on the right side of the
equation. All units accept the one desired are cancelled on the left side of the equation by ensuring that they
appear as both numerator and denominator terms. Unit conversions are an integral part of the equation.
For the example problem, the equation can be written as follows:






1000 !M
C
i
= 5 mM MgCl
2
x = 5000 !M MgCl
2

1 !M
1000 !M x !L
5 mM MgCl
2
x x = 100 !M MgCl
2

1mM 200 !L
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Student Guide
Fall 2012
6

Notice that the equation begins with the initial stock concentration and describes what must be done to that stock
solution to arrive at the desired final concentration on the right side of the equals sign. It asks how many !L (x !L)
of 5 mM MgCl
2
should be placed in a total volume of 200 !L to give a final concentration of 100 !M MgCl
2
. The mM
term is converted to !M within the equation by using the conversion factor 1000 !M/1 mM. All units on the left side
of the equation cancel accept for !M.

Solving for x yields:










All terms cancel and the solution to the problem is revealed to be 4. Since x was originally associated with !L in the
initial equation, you must add 4 !L of 5 mM MgCl
2
to a 200 !L reaction to bring that reaction to 100 !M MgCl
2
.

(5000 !M MgCl
2
)(x)
= 100 !M MgCl
2

200

(200)(100 !M MgCl
2
) 20000
x = = = 4
5000 !M MgCl
2
5000



PCR Optimization
Student Guide
Fall 2012
7

Experiment Overview

Acetylcholine is one of the more thoroughly studied neurotransmitters. During transmission of a nerve impulse
signal, acetylcholine is broken down by the enzyme acetylcholinesterase. The gene for acetylcholinesterase
(abbreviated AChE) is large. It is thousands of base pairs in length. You will determine the optimal concentrations
of primer and magnesium required to amplify, by PCR, a 553 bp segment of the AChE gene as found in the cow
genome. Your class will perform two titration experiments; an increasing amount of either of two reagents will be
added to a series of reaction tubes. In the magnesium titration experiment, the magnesium chloride concentration
will be varied from 0.5 mM to 10 mM. In another set of reactions, the primer concentration will be varied from 0.05
!M to 2 !M.

You will first calculate the volume of each reagent needed in a PCR reaction. In the magnesium titration reactions,
you will keep the primer concentration constant as you vary the magnesium concentration. Likewise, in the primer
titration reactions, you will keep the magnesium chloride concentration constant as you vary the primer
concentration. Therefore, these calculations have been divided into separate sections. After you finish the
calculations, you will need to prepare a master mix, a solution containing all the components for PCR amplification
(with the exception of template and the one component being optimized). Once prepared, aliquots of the master
mix will be distributed equally into 7 reaction tubes. Into each of those 7 tubes will be added a different amount of
the one component you have been assigned to vary (either magnesium chloride or primer). Although water is used
in the preparation of the master mix, in order to keep reaction volumes constant, more will need to be added to
reactions containing less of the varied component. Template DNA will be added as the last component to each
tube. The final volume of each reaction should be 50 !L.




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Student Guide
Fall 2012
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Calculations Part 1: Overview of PCR Reaction Components

As you work through your calculations, the formula (C
i
)(V
i
) = (C
f
)(V
f
) may prove useful.
C
i
= initial concentration C
f
= final concentration

V
i
= initial volume V
f
= final volume
Initial refers to the concentration/volume of the stock reagents and final refers to the desired
concentration/volume.

1. Primer Mix. Each team will prepare a primer mix. The forward and reverse AChE primers are at a
concentration of 50 !M each. You will prepare a primer mix by combining 8 !L of each of the two primers to
give a total combined volume of 16 !L. What is the new concentration of each primer?

C
i
= 50 !M C
f
= x (8 !L)(50 !M) = (x)(16 !L)
V
i
= 8 !L V
f
= 16 !L x = 25 !M

Concentration of each primer in the primer mix = __25 !M_

2. Template DNA. Template DNA is always added last to a PCR reaction. You will be provided with a stock of
Calf Thymus DNA at a concentration of 20 !g DNA/mL. What volume of this template DNA stock is required to
deliver 200 ng to a 50 !L reaction? Hint: 200 ng is an amount, not a concentration.

C
i
= 20 !g/mL x mL/1000 !L x 1000 ng/!g = 20 ng/!L
C
f
= 200 ng/50 !L = 4 ng/!L
V
i
= x (20 ng/!L)(x) = (4 ng/!L)(50 !L)
V
f
= 50 !L x = 10 !L

Into a single 50 !L reaction, add ___10___ !L of 20 !g DNA/mL template stock to give 200 ng of
DNA in that tube.

Note: The following three reagents will be added as components to the master mix made by each team. The
problems below are for a single reaction. The volume of the master mix you prepare will be enough for 8 reactions.
Although you may calculate, for a single reaction, that you may need a volume of a reagent below that of your
pipettes accuracy limit, remember that you will be multiplying this number by 8 later in the protocol when you
prepare the master mix.

3. PCR Buffer. You will be supplied with a stock solution of PCR buffer having a concentration of 10X. In a 50 !L
reaction, you desire a buffer concentration of 1X. To a single 50 !L, how much 10X PCR buffer should be
added to give a final concentration of 1X PCR buffer?

C
i
= 10X C
f
= 1X (10X)(x) = (1X)(50 !L)
V
i
= x V
f
= 50 !L x = 5 !L

To a single 50 !L reaction, add __5 __ !L of 10X PCR buffer to give a concentration of 1X PCR
buffer.

4. dNTPs. You will be provided with a stock solution of dNTPs having a total dNTP concentration of 10 mM. In
this stock solution, the 4 dNTPs are in equal concentrations. Each individual dNTP (dATP, dCTP, dGTP, and
dTTP), therefore, is at a concentration of 2.5 mM (10 mM dNTP/4 individual dNTPs = 2.5 mM each dNTP). In
each 50 !L reaction you will prepare, you need each individual dNTP at a concentration of 200 !M. How much
of the 10 mM dNTP stock solution should be added to each reaction to give this desired individual dNTP
concentration?


C
i
= 2.5 mM x 1000 !M / mM = 2500 !M
C
f
= 200 !M
V
i
= x (2500 !M)(x) = (200 !M)(50 !L)
V
f
= 50 !L x = 4 !L
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Student Guide
Fall 2012
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To a single 50 !L reaction, add ___4___ !L of the 10 mM dNTP stock solution to give a final
concentration of 200 !M for each dNTP.

5. AmpliTaq DNA Polymerase. You will be given a stock solution of AmpliTaq DNA Polymerase having a
concentration of 5 Units/!L. You will need to place 1.25 Units of this enzyme into each 50 !L reaction. How
much of the stock should be added to each tube? Hint: 1.25 Units is an amount, not a concentration. If you
calculate that you will need 12.5 !L of AmpliTaq, then you should try another approach.

C
i
= 5 Units/!L
C
f
= 1.25 Units/50 !L = 0.025 Units / !L
V
i
= x (5 Units/!L)(x) = (0.025 Units/!L)(50 !L)
V
f
= 50 !L x = 0.25 !L

Into a single 50 !L reaction, add ___0.25_ !L of 5 Units/!L AmpliTaq to place 1.25 Units into the
reaction.


PCR Optimization
Student Guide
Fall 2012
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Calculations Part 2: Varying the Magnesium Chloride Concentration

6. AChE Primers. You have previously determined the concentration of each primer in the primer mix to be
___25____ !M. In the MgCl
2
titration series of reactions, you require a concentration of 0.5 !M for each primer
in each 50 !L reaction. How much of the primer mix should be added to each tube to achieve this
concentration?

C
i
= 25 !M C
f
= 0.5 !M (25 !M)(x) = (0.5 !M)(50 !L)
V
i
= x V
f
= 50 !L x = 1 !L

Into a single 50 !L reaction, add ____1___ !L of primer mix to give a final concentration of 0.5 !M
for each primer.

Magnesium chloride (MgCl
2
) will be added to a series of PCR tubes in concentrations of 0.5 mM, 1 mM, 2 mM, 3.5
mM, 5 mM, and 10 mM. The stock solution of MgCl
2
you will be given has a concentration of 25 mM. In the spaces
provided, calculate the volumes of 25 mM MgCl
2
stock solution required for this reaction set to bring each 50 !L
reaction to its desired magnesium concentration.

7. For a final concentration of 0.5 mM MgCl
2
:

C
i
= 25 mM
C
f
= 0.5 mM
V
i
= x (25 mM)(x) = (0.5 mM)(50 !L)
V
f
= 50 !L x = 1 !L

Into a single 50 !L reaction, add ___1____ !L of the 25 mM MgCl
2
stock solution to yield a final
concentration of 0.5 mM magnesium.

a) Is this a practical amount to deliver with a micropipet? Why or why not?
No. p20 micropipets can not deliver 1 !L volumes accurately.
The pipets used in this class cannot deliver volumes much smaller than 1 !L with reliable accuracy. Therefore, it
will be necessary to prepare a diluted magnesium chloride sample from which more accurate volumes can be
dispensed. You will dilute 5 !L of 25 mM MgCl
2
into 5 !L of sterile water.

b) What type of dilution does this represent (i.e., 1/2, 1/10, 1/20, 1/100, 1/1000)?
1/2
c) What will be the concentration of this new diluted magnesium chloride stock?
25 mM / 2 = 12.5 mM
d) From this dilution, how many microliters should be added to a 50 !L reaction to bring the final concentration of
magnesium chloride to 0.5 mM?
C
i
= 12.5 mM
C
f
= 0.5 mM
V
i
= x (12.5 mM)(x) = (0.5 mM)(50 !L)
V
f
= 50 !L x = 2 !L

Into a single 50 !L reaction, add ___2___ !L of the diluted MgCl
2
to yield a final concentration of
0.5 mM of MgCl
2
.

8. For a final concentration of 1 mM MgCl
2
(using the 25 mM MgCl
2
stock):

C
i
= 25 mM
C
f
= 1 mM
V
i
= x (25 mM)(x) = (1 mM)(50 !L)
V
f
= 50 !L x = 2 !L

Into a single 50 !L reaction, add ____2___ !L of the 25 mM MgCl
2
stock solution to yield a final
concentration of 1 mM magnesium.
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Student Guide
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9. For a final concentration of 2 mM MgCl
2
:

C
i
= 25 mM
C
f
= 2 mM
V
i
= x (25 mM)(x) = (2 mM)(50 !L)
V
f
= 50 !L x = 4 !L

Into a single 50 !L reaction, add ___4____ !L of the 25 mM MgCl
2
stock solution to yield a final
concentration of 2 mM magnesium.

10. For a final concentration of 3.5 mM MgCl
2
:

C
i
= 25 mM
C
f
= 3.5 mM
V
i
= x (25 mM)(x) = (3.5 mM)(50 !L)
V
f
= 50 !L x = 7 !L

Into a single 50 !L reaction, add ____7___ !L of the 25 mM MgCl
2
stock solution to yield a final
concentration of 3.5 mM magnesium.

11. For a final concentration of 5 mM MgCl
2
:

C
i
= 25 mM
C
f
= 5 mM
V
i
= x (25 mM)(x) = (5 mM)(50 !L)
V
f
= 50 !L x = 10 !L


Into a single 50 !L reaction, add ___10___ !L of the 25 mM MgCl
2
stock solution to yield a final
concentration of 5 mM magnesium.


12. For a final concentration of 10 mM MgCl
2
:

C
i
= 25 mM
C
f
= 10 mM
V
i
= x (25 mM)(x) = (10 mM)(50 !L)
V
f
= 50 !L x = 20 !L

Into a single 50 !L reaction, add ___20___ !L of the 25 mM MgCl
2
stock solution to yield a final
concentration of 10 mM magnesium.



Calculating the volumes of water to use for each reaction in the magnesium titration series to keep all
reactions at 50 !L:

With the exception of MgCl
2
, all reagents should be at equivalent concentrations in the series of tubes you will be
preparing. Hopefully this is obvious to you at this point since the variable you are inspecting, MgCl
2
, should be the
only reagent that changes.

Use Table 4 below to summarize what will be added to each of the tubes.




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Student Guide
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NOTE: The volumes to be entered below are for one single tube.

Volume of Reaction Components for Magnesium Titration

Reaction
Component
Tube 1M
0.5 mM
MgCl
2

Tube 2M
1 mM
MgCl
2

Tube 3M
2 mM
MgCl
2

Tube 4M
3.5 mM
MgCl
2

Tube 5M
5 mM
MgCl
2

Tube 6M
10 mM
MgCl
2

Tube 7M
No DNA
Control
(1 mM
MgCl
2
)
10X PCR
Buffer

5 !L

5 !L

5 !L

5 !L

5 !L

5 !L

5 !L
10 mM
dNTP stock

4 !L

4 !L

4 !L

4 !L

4 !L

4 !L

4 !L
AmpliTaq
polymerase

0.25 !L

0.25 !L

0.25 !L

0.25 !L

0.25 !L

0.25 !L

0.25 !L
20 !g/mL
Template
DNA

10 !L

10 !L

10 !L

10 !L

10 !L

10 !L

0 !L
AChE primer
mix

1 !L

1 !L

1 !L

1 !L

1 !L

1 !L

1 !L

MgCl
2




__2_ !L
1/2 dil of
25 mM
MgCl
2



__2_ !L
25 mM
MgCl
2



__4_ !L
25 mM
MgCl
2



__7_ !L
25 mM
MgCl
2



__10_!L
25 mM
MgCl
2



__20_!L
25 mM
MgCl
2



__2_ !L
25 mM
MgCl
2


Volume of
above
components

22.25 !L

22.25 !L

24.25 !L

27.25 !L

30.25 !L

40.25 !L

12.25 !L
Table 4. Summary of Reagents Added

Notice that the volume of above components row has a variety of values. That means, at this point, that the final
volume in your tube will all be different (except Tubes 1M and 2M). So a wise person may ask, How can all the
reagents added be at the same concentration if the final volumes are different? And they wouldnt be. So lets take
one more thing into consideration and then make the final volumes identical so that the only concentration that is
changing is the MgCl
2
. The final volume of each of the reactions will be made identical by the addition of
water.

Preparing a Master Mix. Remember that the volumes you have entered in Table 4 were all for each tube. That
means you have to add 5 !L of PCR buffer to each of 7 tubes. Then add 4 !L of 10 mM dNTP stock to 7 tubes and
so on. That is a lot of pipetting and each time you pipet you can introduce some error in the measurement. So
we will use a way to minimize the pipetting. We will make a Master Mix. The Master Mix for this experiment should
contain all the reagents needed for the PCR except for the one component being titrated (in this case, magnesium)
and the template DNA (which should always be added last to a PCR). The Master Mix will also contain the
smallest amount of water any of the 7 tubes will need. Once prepared, an aliquot of the Master Mix will be
delivered to each reaction tube. Then, different amounts of MgCl
2
will be added to each tube, and additional water
will be added to each reaction such that the final volume will be 50 !L. Template DNA will be added as the final
step prior to thermal cycling.
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Student Guide
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Even though you have seven tubes where each tube is a trial, you will make a Master Mix for 8 reactions to ensure
that enough Master Mix is available. See last paragraph of this page.

Calculating Master Mix Volumes. Water will need to be part of a Master Mix so that concentrated salts in the
reaction are diluted. This will be the least amount of water every tube will contain. Later we will add additional
water to ensure proper dilutions and volumes.

A. What is the largest volume of MgCl
2
being added to any one reaction (see Table 4)?


__20__ !L


B. For the reaction corresponding to this MgCl
2
amount, what is the volume of water required to bring that reaction
to 50 !L?
_9.75__ !L



At least this amount of water will be contained in each reaction you will prepare. This amount of water will be used
in calculating a Master Mix. Enter this volume in the first box of Column 3 of Table 5.

The volumes of reagents required to prepare a Master Mix can now be calculated using Table 5.To ensure that
enough Master Mix is available, calculate the total volumes of each reagent needed assuming 8 reactions must be
prepared. The amount of water to use in Column 3 was determined on the previous page. You will find the other
reagent volumes listed in Table 4.


Master Mix Reagent Components (Magnesium Titration)
Column 1 2 3 4 5
Reaction
Component
Stock
Solution
Final Conc.
Amount for
1 Reaction
(in !L)
Number of
Reactions
Master Mix
Total Volume
(Column 3 X
Column 4)
water 9.75 !L 8 78 !L
PCR Buffer 10X 1X 5 !L 8 40 !L
dNTPs
2.5 mM
each
200 !M
each
4 !L 8 32 !L
AmpliTaq 5 Units/!L 1.25 Units 0.25 !L 8 2 !L
Primer Mix 25 !M
0.5 !M
each
1 !L 8 8 !L
Total 20 !L
Table 5. Preparing the Master Mix for the magnesium titration experiment.

Additional Water to Add to Each Reaction: Use Table 6 to calculate how much additional water (in addition to
the 9.75 !L added to the Master Mix) should be added to each reaction tube so that the total volume of MgCl
2

plus water is equal to the largest single volume of MgCl
2
being added. No additional water will need to be added to
the reaction containing the largest volume of MgCl
2
. For example, for Tube 1M the volume of diluted MgCl
2

solution to add is 2 !L. The largest single volume of MgCl
2
being added is 20 !L. Therefore, 18 !L of additional
water needs to be added to tube 1M.

Important Notice: Please note that for tube 1M you are adding 2 !L of diluted MgCl
2
. ALL OTHER
TUBES, 2M7M RECEIVE THE STOCK SOLUTION (25 Mm MgCl
2
).

PCR Optimization
Student Guide
Fall 2012
14

Additional Water to Add to Magnesium Titration Reactions

Reaction
Number

MgCl
2
Concentration

Volume of MgCl
2

solution to add
(in !L)

Volume of additional water
to add to each reaction
(in !L)
1M 0.5 mM 2 !L of diluted MgCl
2
18 !L
2M 1 mM 2 !L 18 !L
3M 2 mM 4 !L 16 !L
4M 3.5 mM 7 !L 13 !L
5M 5 mM 10 !L 10 !L
6M 10 mM 20 !L 0 !L
7M
No DNA Control,
1 mM
2!L 18 !L
Table 6. Volumes of Additional Water to Add to Each Reaction in the Magnesium Series.

PCR Optimization
Student Guide
Fall 2012
15

Calculations Part 3: Varying the Primer Concentration

13. MgCl
2
: The MgCl
2
stock solution you will be given is at a concentration of 25 mM. In the primer titration series
of reactions, you require a final MgCl
2
concentration of 1 mM in each tube. How many microliters of the 25 mM
MgCl
2
stock solution should be added to each reaction to give a final magnesium chloride concentration of 1
mM?

C
i
= 25 mM
C
f
= 1 mM
V
i
= x (25 mM)(x) = (1 mM)(50 !L)
V
f
= 50 !L x = 2 !L

Into a single 50 !L reaction, add ____2___ !L of 25 mM MgCl
2
to give a final concentration of 1
mM magnesium.

Varying amounts of primer mix will be added to a series of PCR tubes at concentrations of 0.05 !M, 0.1 !M, 0.2
!M, 0.5 !M, 1 !M, and 2 !M for each primer. You have previously calculated the concentration of each primer in
the primer mix to be _____ !M. In the spaces provided, calculate the volumes of primer mix required for this
reaction set to bring each 50 !L reaction to its desired primer concentration.

14. For a final concentration of 0.05 !M each primer:
a) To bring the primer concentration of a 50 !L reaction to 0.05 !M for each primer, what volume of primer mix
should be added?

C
i
= 25 !M
C
f
= 0.05 !M
V
i
= x (25 !M)(x) = (0.05 !M)(50 !L)
V
f
= 50 !L x = 0.1 !L

Into a single 50 !L reaction, add __0.1__ !L of the primer mix to yield a final concentration of 0.05 !M
for each primer.

15. Is this a practical amount to deliver with a micropipette? Why or why not?
No. p20 micropipettes can not deliver 0.1 !L volumes accurately.

The pipets used in this class cannot deliver volumes much smaller than 1 !L with reliable accuracy. Therefore, it
will be necessary to prepare a diluted primer sample from which more accurate volumes can be dispensed. You will
dilute 4 !L of the primer mix into 76 !L of water.

b) What type of dilution does this represent (i.e., 1/2, 1/10, 1/20, 1/100, 1/1000)?
4 !L/80 !L total volume = 1/20 dilution
c) What will be the concentration of this new diluted primer stock? 1.25 !M

C
i
= 25 !M C
f
= x (25 !M)(4 !L) = (x)(80 !L)
V
i
= 4 V
f
= 80 !L x = 1.25 !M

d) From this dilution, how many microliters should be added to a 50 !L reaction to bring the primer concentration
in that reaction to 0.05 !M for each primer?

C
i
= 1.25 !M
C
f
= 0.05 !M
V
i
= x (1.25 !M)(x) = (0.05 !M)(50 !L)
V
f
= 50 !L x = 2 !L

Into a single 50 !L reaction, add ___2___ !L of the diluted primer mix to yield a final concentration
of 0.05 !M for each primer.

PCR Optimization
Student Guide
Fall 2012
16

15. For a final concentration of 0.1 !M each primer: You will use your diluted primer mix for this reaction as well.
How many !L of the diluted primer mix should be added to a 50 !L reaction to bring the primer concentration to
0.1 !M for each primer?

C
i
= 1.25 !M
C
f
= 0.1 !M
V
i
= x (1.25 !M)(x) = (0.1 !M)(50 !L)
V
f
= 50 !L x = 4 !L

Into a single 50 !L reaction, add ___4___ !L of the diluted primer mix to yield a final concentration
of 0.1 !M for each primer.

16. For a final concentration of 0.2 !M each primer: You will use your diluted primer mix for this reaction too.
How many !L of the diluted primer mix should be added to a 50 !L reaction to bring the primer concentration to
0.2 !M for each primer?

C
i
= 1.25 !M
C
f
= 0.2 !M
V
i
= x (1.25 !M)(x) = (0.2 !M)(50 !L)
V
f
= 50 !L x = 8 !L

Into a single 50 !L reaction, add ___8___ !L of the diluted primer mix to yield a final concentration
of 0.2 !M for each primer.

17. For a final concentration of 0.5 !M each primer: How many !L of the diluted primer mix should be added to
a 50 !L reaction to bring the primer concentration to 0.5 !M for each primer?

C
i
= 1.25 !M
C
f
= 0.5 !M
V
i
= x (1.25 !M)(x) = (0.5 !M)(50 !L)
V
f
= 50 !L x = 20 !L

Into a single 50 !L reaction, add __20___ !L of the diluted primer mix to yield a final concentration
of 0.5 !M for each primer.

NOTE: For this next reaction and all subsequent reactions requiring even higher primer concentrations, you will
use the original primer mix. This will ensure that the volume of primer you add will not result in a final reaction
volume that exceeds 50 !L.

18. For a final concentration of 1 !M each primer: How many !L of the original primer mix should be added to a
50 !L reaction to bring the primer concentration to 1 !M for each primer?

C
i
= 25 !M
C
f
= 1 !M
V
i
= x (25 !M)(x) = (1 !M)(50 !L)
V
f
= 50 !L x = 2 !L

Into a single 50 !L reaction, add ___2___ !L of the original primer mix to yield a final concentration
of 1 !M for each primer.

19. For a final concentration of 2 !M each primer: How many !L of the original primer mix should be added to a
50 !L reaction to bring the primer concentration to 2 !M for each primer?

C
i
= 25 !M
C
f
= 2 !M
V
i
= x (25 !M)(x) = (2 !M)(50 !L)
V
f
= 50 !L x = 4 !L
PCR Optimization
Student Guide
Fall 2012
17


Into a single 50 !L reaction, add ___4___ !L of the original primer mix to yield a final concentration
of 2 !M for each primer.

Calculating the volumes of water to use for each reaction in the primer titration series to keep all
reactions at 50 !L.

With the exception of primers, all reagents should be at equivalent concentrations in the series of tubes you will be
preparing. This hopefully is obvious to you at this point since the variable you are inspecting, primers, should be
the only reagent that changes.

Use Table 7 below to summarize what will be added to each of the tubes.

NOTE: The volumes to be entered below are for one single tube.

Volume of Reaction Components by Primer Concentration

Reaction
Component
Tube 1P

0.05 !M
Primer
Tube 2P

0.1 !M
Primer
Tube 3P

0.2 !M
Primer
Tube 4P

0.5 !M
Primer
Tube 5P

1 !M
Primer
Tube 6P

2 !M
Primer
Tube 7P
No DNA
Control
(0.5 !M
Primer)
10X PCR
Buffer

5 !L

5 !L

5 !L

5 !L

5 !L

5 !L

5 !L
10 mM dNTP
stock

4 !L

4 !L

4 !L

4 !L

4 !L

4 !L

4 !L
AmpliTaq
polymerase

0.25 !L

0.25 !L

0.25 !L

0.25 !L

0.25 !L

0.25 !L

0.25 !L
20 !g/mL
Template
DNA

10 !L

10 !L

10 !L

10 !L

10 !L

10 !L

0 !L
AChE primer
mix

__2_ !L
Diluted
Primer
Mix

__4_ !L
Diluted
Primer
Mix

__8_ !L
Diluted
Primer
Mix

_20_ !L
Diluted
Primer
Mix

__2_ !L
Primer
Mix

__4_ !L
Primer
Mix

_20_ !L
Diluted
Primer
Mix

25 mM
MgCl
2



2 !L

2 !L

2 !L

2 !L

2 !L

2 !L

2 !L
Volume of
above
components

23.25 !L

25.25 !L

29.25 !L

41.25 !L

23.25 !L

25.25 !L

31.25 !L
Table 7. Summary of Reagents Added.

Notice that the volume of above components row has a variety of values. That means, at this point, that the final
volume in your tube will all be different (except Tubes 1P and 5P, and 2P and 6P). So a wise person may ask,
How can all the reagents added be at the same concentration if the final volumes are different? And they wouldnt
be. So lets take one more thing into consideration and then make the final volumes identical so that the only
concentration that is changing is the primers. The final volume of each of the reactions will be made identical
by the addition of water.

PCR Optimization
Student Guide
Fall 2012
18

Preparing a Master Mix. Remember that the volumes you have entered in Table 7 were all for each tube. That
means you have to add 5 !L of PCR buffer to each of 7 tubes. Then add 4 !L of 10 mM dNTP stock to 7 tubes and
so on. That is a lot of pipetting and each time you pipet you can introduce some error in the measurement. So
we will use a way to minimize the pipetting. We will make a Master Mix. The Master Mix for this experiment should
contain all the reagents needed for the PCR except for the one component being titrated (in this case, primer) and
the template DNA (which should always be added last to a PCR). The Master Mix will also contain the smallest
amount of water any of the 7 tubes will need. Once prepared, an aliquot of the Master Mix will be delivered to
each reaction tube. Then, different amounts of primers will be added to each tube, and additional water will be
added to each reaction such that the final volume will be 50 !L. Template DNA will be added as the final step prior
to thermal cycling.

Even though you have seven tubes where each tube is a trial, you will make a Master Mix for 8 reactions to ensure
that enough Master Mix is available. See last paragraph of this page.

Calculating Master Mix Volumes. Water will need to be part of a Master Mix so that concentrated salts in the
reaction are diluted. This will be the least amount of water every tube will contain. Later we will add additional
water to ensure proper dilutions and volumes.

A. What is the largest volume of primer being added to any one reaction (see Table 7)?

__20__ !L

B. For the reaction corresponding to this primer amount, what is the volume of water required to bring that reaction
to 50 !L?
_8.75__ !L

At least this amount of water will be contained in each reaction you will prepare. This amount of water will be used
in calculating a Master Mix. Enter this volume in the first box of Column 3 of Table 8.

The volumes of reagents required to prepare a Master Mix can now be calculated using Table 8. To ensure that
enough Master Mix is available, calculate the total volumes of each of reagent needed in Table 8, assuming 8
reactions will be prepared.

Use Table 7 to fill in other volumes for the other reagents in Table 8.

Master Mix Reagent Components (Primer Titration)
Column 1 2 3 4 5
Reaction
Component
Stock
Solution
Final
Conc.
Amount for
1 Reaction
(in !L)
Number of
Reactions
Master Mix Total
Volume
(Column 3 X
Column 4)
water
8.75 !L 8 70 !L
PCR Buffer
10X 1X 5 !L 8 40 !L
dNTPs 2.5 mM
each
200 !M
each
4 !L 8 32 !L
AmpliTaq
5 Units/!L 1.25 Units 0.25 !L 8 2 !L
MgCl
2

25 mM 1 mM 2 !L 8 16 !L
Total
20 !L
Table 8. Preparing the Master Mix for the Primer Titration Experiment.





PCR Optimization
Student Guide
Fall 2012
19

Additional Water to Add to Each Reaction: Use Table 9 below to calculate how much additional water (a volume
in addition to the 8.75 !L added to the Master Mix) should be added to each reaction tube so that the total volume
of primer plus water is equal to the largest single volume of primer being added. No additional water will need to be
added to the reaction containing the largest volume of primer. For example, for Tube 1P the volume of diluted
primer mix to add is 2 !L. The largest single volume of primer being added is 20 !L. Therefore, 18 !L of
additional water needs to be added to tube 1P.

Important Notice: Please note that for tube 1P4P AND 7P you are adding diluted Primer Mix. ALL OTHER
TUBES, 5P6P RECEIVE THE PRIMER MIX SOLUTION.


Reaction
Number

Primer
Concentration

Primer
Source

Volume of primer to
Add (in !L)
Volume of
additional water to
add (in !L)
1P 0.05 !M Diluted Primer Mix 2 !L 18 !L
2P 0.1 !M Diluted Primer Mix 4 !L 16 !L
3P 0.2 !M Diluted Primer Mix 8 !L 12 !L
4P 0.5 !M Diluted Primer Mix 20 !L 0 !L
5P 1 !M Primer Mix 2 !L 18 !L
6P 2 !M Primer Mix 4 !L 16 !L
7P
No DNA Control,
0.5 !M
Diluted Primer Mix 20 !L 0 !L
Table 9. Volumes of additional water to add to each reaction in the primer titration series to make them
equal in volume to that of the sample receiving the largest volume of primer.


PCR Optimization
Student Guide
Fall 2012
20

Laboratory Exercise

Your teacher will divide you into teams to perform either the magnesium or the primer optimization protocol. In
performing this experiment, keep these laboratory practices in mind:

Important Laboratory Practices
a. Add reagents to the bottom of the reaction
tube, not to its side.
b. Add each additional reagent directly into
previously-added reagent.
c. Do non pipette up and down to mix, as
this introduces error. This should only be
done only when resuspeding the cell pellet
and not to mix reagents.
d. Make sure contents are all settled into the
bottom of the tube and not on the side or
cap of tube. A quick spin may be needed
to bring contents down.



a. Pipet slowly to prevent contaminating the
pipette barrel.
b. Change pipette tips between each delivery.
c. Change the tip even if it is the same reagent
being delivered between tubes. Change tip
every time the pipette is used!



Keep reagents on ice.


Check the box next to each step as you complete it.

PCR Optimization
Student Guide
Fall 2012
21


Varying Magnesium Concentration
1. Label a 1.5 mL tube "PM," for Primer Mix.

!
2. Into your "PM" tube, add 8 !L of each of the two
AChE primers (forward and reverse). Keep this
tube on ice.

!
3. Label a new 1.5 mL tube "1/2 Mg," for 1/2 dilution
of MgCl
2
.


!
4. Into your "1/2 Mg" tube, add 5 !L of sterile water
and 5 !L of 25 mM MgCl
2
. Keep this tube on ice.




!
5. Label another new 1.5 mL tube "MM," for Master
Mix.

!
PCR Optimization
Student Guide
Fall 2012
22

6. Prepare the Master Mix. Into the "MM" tube, add
each reaction component according to the volumes
calculated for Column 5, Table 5. Add the water to
the MM tube first. Keep the Master Mix and all
Master Mix components on ice. Template DNA is
not added to the master mix; it will be added
individually to each reaction later.

Note: Slowly pipet up and down to mix the reagents
after each addition.
water = 78 !L PCR Buffer = 40 !L
dNTP Mix = 32 !L AmpliTaq = 2 !L
PM = 8 !L






!
7. Mark seven 0.2 mL tubes "1M" through "7M". Also, label each tube with your initials.





!
8. Gently flick the Master Mix tube with your finger to
mix the solution.












!
9. Making sure to use a balance tube, spin the Master
Mix tube for 5 seconds in a microcentrifuge.










!
PCR Optimization
Student Guide
Fall 2012
23

10. To each tube, add the appropriate combination of MgCl
2
and water (see Table 6). To each tube except "7M,"
also add the volume of template DNA as determined previously. To tube "7M" (the Control), add a volume of
additional water equal to the volume of DNA being added to each tube 1M through 6M. [_18_ !L water for
tube 7M from Table 6 + __10_ !L water equivalent to the amount of template DNA being added to each
reaction = __28_ !L total water for tube 7M.]

Tube

Reagent
1M
(0.5 mM
Mg)
2M
(1 mM
Mg)
3M
(2 mM
Mg)
4M
(3.5 mM
Mg)
5M
(5 mM
Mg)
6M
(10 mM
Mg)
7M
(1 mM
Mg)
Water 18 !L 18 !L 16 !L 13 !L 10 !L 0 !L 28 !L
1:2 dil. Mg 2 !L - - - - - -
25 mM Mg - 2 !L 4 !L 7 !L 10 !L 20 !L 2 !L
Template 10 !L 10 !L 10 !L 10 !L 10 !L 10 !L 0 !L

!
11. Place your reactions into the thermal cycler and
record the location of your tubes on the grid
provided by your teacher.












!
The cycling protocol for amplification using these AChE
primers is as follows:

95C10 minutes
95C1 minute
54C1 minute 35 cycles
72C1 minute
72C10 minutes
4Chold, " infinity



!
! " # $ % & '
( !!"# )")
* !+"'
, &''' -#+%
PCR Optimization
Student Guide
Fall 2012
24


Varying Primer Concentration
1. Label a 1.5 mL tube "PM" for Primer Mix.

!
2. Into your "PM" tube, add 8 !L of each of the two
AChE primers (forward and reverse). Keep this
tube on ice.

!
3. Label a new 1.5 mL tube 1/20 PM," for a 1/20
dilution of your Primer Mix.

!
4. Into the "1/20 PM" tube, add 76 !L of sterile
distilled water and 4 !L of Primer Mix (from Step 2).
Keep this tube on ice.

!
5. Label a new 1.5 mL tube "MM," for Master Mix.

!
PCR Optimization
Student Guide
Fall 2012
25

6. Prepare the Master Mix. Into the "MM" tube, add
each reaction component according to the volumes
calculated for Column 5, Table 8. Add the water to
the MM tube first. Keep the Master Mix and all
Master Mix components on ice. Template DNA is
not added to the master mix; it will be added
individually to each reaction later.

NOTE: Slowly pipet up and down to mix the reagents
after each addition.

Water = 70 !L PCR Buffer = 40 !L
dNTP Mix = 32 !L AmpliTaq = 2 !L
25 mM MgCl
2
= 16 !L

!
7. Mark seven 0.2 mL tubes "1P" through "7P". Label each tube with your initials as well.


!
8. Gently flick the Master Mix tube with your finger to
mix the solution.













!
9. Making sure to use a balance tube, spin the Master
Mix tube for 5 seconds in a microcentrifuge.











!
PCR Optimization
Student Guide
Fall 2012
26

10. Pipet into each of your 7 labeled 0.2 mL tubes the volume of Master Mix as calculated in Table 8, Total for
Column 3. 20 !L into each tube


!
11. To each tube, add the appropriate combination of Primer Mix and water (see Table 9). To each tube except
"7P," also add the volume of template DNA as determined previously. To tube "7P" (the Control), add a
volume of additional water equal to the volume of template added to each tube 1P through 6P. [__0 _ !L
water for tube 7P from Table 9 + __10_ !L water equivalent to the amount of template DNA being added
to each tube = _10__ !L total water for tube 7P.]

Tube

Reagent
1P
(0.05 !M
Primer)
2P
(0.1 !M
Primer)
3P
(0.2 !M
Primer)
4P
(0.5 !M
Primer)
5P
(1 !M
Primer)
6P
(2 !M
Primer)
7P
(0.5 !M
Primer)
Water 18 !L 16 !L 12 !L 0 !L 18 !L 16 !L 10 !L
1/20 PM 2 !L 4 !L 8 !L 20 !L - - 20 !L
PM - - - - 2 !L 4 !L -
Template 10 !L 10 !L 10 !L 10 !L 10 !L 10 !L 0 !L
!
12. Place your reactions into the thermal cycler and
record the location of your tubes on the grid
provided by your teacher.












!
The cycling protocol for amplification using these AChE
primers is as follows:

95C10 minutes
95C1 minute
54C1 minute 35 cycles
72C1 minute
72C10 minutes
4Chold, " infinity
!
! " # $ % & '
( !!"# )")
* !+"'
, &''' -#+%
PCR Optimization
Student Guide
Fall 2012
27

Agarose Gel Electrophoresis

To determine whether or not the AChE PCR product amplified, you will need to visualize the products of your
amplification. This will be done using a process called gel electrophoresis in which electric current forces the
migration of DNA fragments through a special gel material. Since DNA is negatively charged, it will migrate in an
electric field towards the positive electrode (Figure 2). When electrophoresed through a gel, shorter fragments of
DNA move at a faster rate than longer ones.

Figure 2. Side view of an
agarose gel showing DNA
loaded into a well and the
direction of DNA fragment
migration during
electrophoresis.


The gel material to be used for this experiment is called agarose, a gelatinous substance derived from a
polysaccharide in red algae. When agarose granules are placed in a buffer solution and heated to boiling
temperatures, they dissolve and the solution becomes clear. A comb is placed in the casting tray to provide a mold
for the gel. The agarose is allowed to cool slightly and is then poured into the casting tray. Within about 15 minutes,
the agarose solidifies into an opaque gel having the look and feel of coconut Jell-O. The gel, in its casting tray, is
placed in a buffer chamber connected to a power supply and running buffer is poured into the chamber until the gel
is completely submerged. The comb can then be withdrawn to form the wells into which your PCR sample will be
loaded.

Loading dye is a colored, viscous liquid containing dyes (making it easy to see) and sucrose, Ficoll, or glycerol
(making it dense). To a small volume of your total PCR reaction, you will add loading dye, mix and then pipet an
aliquot of the mixture into one of the wells of your agarose gel. When all wells have been loaded with sample, you
will switch on the power supply. The samples should be allowed to electrophorese until the dye front (either yellow
or blue, depending on the dye used) is 1 to 2 cm from the bottom of the gel. The gel can then be moved, stained
and photographed.

Calculations for Preparing 2% Agarose Gel
You will need a 2%, mass/volume agarose gel for electrophoresis of your PCR products. If your agarose gel
casting trays holds 50 mL, then how much agarose and buffer would you need? The definition of m/v % in biology
is grams (mass) / 100 mL (volume). Therefore, for 2% agarose, it will be 2 g /100 mL buffer.

Step 1: Calculate the mass of agarose needed for 50 mL total volume of agarose solution.






Step 2: Calculate the amount of buffer needed to bring the agarose solution to 50 mL. By standard definition, 1
gram of H
2
O = 1 mL of H
2
O. The amount of buffer for the 2% agarose solution will be 49 mL (50 mL 1 mL (1 gram
of agarose)).



2 g X g
= X = 1 gram
100 ml 50 ml


PCR Optimization
Student Guide
Fall 2012
28


Electrophoresis of Amplified DNA
1. Retrieve your PCR tube and place it in a balanced
configuration in a microcentrifuge. Spin it briefly (10
seconds) to bring the liquid to the bottom of the reaction
tube.

Note: Make sure the centrifuge adapters are in place before
putting the tiny PCR tube into the centrifuge rotor.



2. Add 5 !L of loading dye to your PCR tube.



3. Carefully load 15 to 20 !L of the DNA/loading dye mixture
into a well in your gel. Make sure you keep track of what
sample is being loaded into each well.

Note: Avoid poking the pipette tip through the bottom of the
gel or spilling sample over the sides of the well. Use a new
tip for each sample.


4. One student (or the instructor) should load 5-10 !L of 100
bp ladder (molecular weight marker) into one of the wells of
each gel.



5. When all samples are loaded, attach the electrodes from the
gel box to the power supply. Have your teacher check your
connections and then electrophorese your samples at 150
Volts for 2540 minutes.




6. After electrophoresis, the gels will be ready to stain and
photograph.



PCR Optimization
Student Guide
Fall 2012
29


Staining and Photographing Agarose Gels

The PCR products separated on your agarose gel are invisible to the naked eye. If you look at your gel in normal
room light, you will not be able to see the amplified products of your reaction. In order to see them, we must stain
the gel with a fluorescent dye called ethidium bromide (EtBr). Molecules of ethidium bromide are flat and can
intercalate, or insert, between adjacent base pairs of double stranded DNA (Figure 3). When this interaction
occurs, they take on a more ordered and regular configuration causing them to fluoresce under ultraviolet light
(UV). Exposing the gel to UV light after staining, allows you to see bright, pinkish-orange bands where there is DNA
(figure 4).


Figure 3. Ethidium bromide
molecules intercalated between
DNA base pairs.








Your teacher may stain your agarose gel and take a photograph for you so that you may analyze your PCR results.
Gel staining is done as follows:
1. Place the agarose gel in a staining tray.
2. Pour enough ethidium bromide (0.5!g/ mL) to cover the gel.
3. Wait 20 minutes.
4. Pour the ethidium bromide solution back into its storage bottle.
5. Pour enough water into the staining tray to cover the gel and wait 5 minutes.
6. Pour the water out of the staining tray into a hazardous waste container and place the stained gel on a
UV light box.
7. Place the camera over the gel and take a photograph.
8. Check with your district on how to dispose of hazardous waste liquid and solids.


CAUTION: Ethidium bromide is considered a carcinogen and neurotoxin. Always wear gloves and
appropriate PPE (personal protective equipment) like safety glasses when handling. Students should
NEVER handle EtBr.

CAUTION: Ultraviolet light can damage your eyes and skin. Always wear protective clothing and UV safety
glasses when using a UV light box.


Figure 4. After staining an agarose gel
with ethidium bromide, DNA bands are
visible upon exposure to UV light.




PCR Optimization
Student Guide
Fall 2012
30

Interpretation of Results

Role of Magnesium

AmpliTaq DNA Polymerase adds bases onto the end of an annealed primer. Magnesium ions interact with the
DNA polymerase enzyme during this process. Magnesium, in fact, is absolutely required for DNA polymerase
activity. Because of the close relationship between these molecules, magnesium is said to be a cofactor of the
polymerase enzyme. PCR amplification of a DNA target would not occur if magnesium were left out of the reaction.

Magnesium is usually supplied to a PCR amplification in the form of magnesium chloride. As this compound is a
salt, in the water environment of a DNA synthesis reaction, it dissociates into the two ions Mg
++
and Cl
-
. Because of
its positive charge, the magnesium ion interacts with negatively charged molecules in the reaction. What are these
molecules? Template DNA is one of them. It has negatively charged phosphate groups running along its backbone.
The primers too are DNA and even though single-stranded, have a sugar-phosphate backbone. This gives them a
negative charge. Of critical importance, however, are the dNTPs. Each carries a total of 4 negatively charged
oxygen molecules attached to the triphosphate group (see Figure below). Relative to the other reaction
components, the dNTPs are in a very high concentration and therefore constitute the predominant species
interacting and binding to the magnesium ions.


Figure 5. Molecular structure of the dNTP, deoxyadenosine triphosphate (dATP). There are four negatively-
charged oxygen molecules surrounding the phosphate atoms. These can interact with magnesium ions.

Consequently, in a PCR experiment, it is necessary to use an amount of MgCl
2
above that of the dNTP
concentration; some magnesium must be left free and available for use by the AmpliTaq DNA Polymerase enzyme.
If magnesium is not available, no extension can occur.



Magnesium and DNA

DNA has an overall negative charge because of the negatively charged oxygen molecules along the two sugar-
phosphate chains of the double helix. Since both chains are negatively charged, they have a natural tendency to
repel each other. In fact, if DNA is placed in water free of any ions, the two strands of DNA are very likely to come
apart. Positive ions such as Na
+
and Mg
++
(found in sodium chloride and magnesium chloride), however, can
interact with the negatively charged DNA strands to mask the forces of repulsion. The higher the salt concentration,
the more likely DNA will remain double-stranded. In addition, at higher salt concentrations, two strands of DNA can
be made to anneal to each other even if there is not perfect complementarity between them. Under conditions of
very high salt concentrations, the double helix structure for some DNA segments can be quite stable, so much so
that an even higher temperature is required to denature it.




PCR Optimization
Student Guide
Fall 2012
31


Magnesium Optimization: Interpreting Results

On an agarose gel, an optimized PCR amplification should give one single, bright band of the desired size. A
poorly-optimized reaction will show the desired band in reduced intensity and the presence of non-specific bands of
different sizes. The AChE primers used in this experiment will produce a 553 bp PCR product.


Questions:
a) Did you obtain the 553 bp band expected for this experiment?



b) Which magnesium chloride concentration seems to be optimal for amplification of this bovine
acetylcholinesterase gene segment?



c) What do you notice in the lane with the lowest magnesium concentration? Why?



d) What do you notice in the lanes with higher magnesium concentrations? Why?



e) What advantage does using a master mix provide the experimenter?


PCR Optimization
Student Guide
Fall 2012
32

Role of Primers

Primers are short, single-stranded DNAs designed to anneal to complementary sequences on opposite strands of
the template DNA flanking the target region. They serve as initiation sites for the addition of bases by AmpliTaq
DNA Polymerase. For successful PCR, primers must be added in molar excess over the amount of target DNA.
This ensures that, following the denaturation step primer/template annealing is favored over template/template re-
annealing.

During PCR, an annealing step is used to allow primer attachment to the template. The temperature chosen for
annealing is based on the melting temperature (T
m
) of the primers. The melting temperature of a particular DNA
molecule is defined as that temperature at which half of it is in double-stranded form and half is in single-stranded
form. Longer DNA molecules, because of the many hydrogen bonds formed between their two strands, have higher
melting temperatures than shorter DNA molecules. Similarly, if two DNA molecules are of the same size but have
different numbers of G and C bases, the molecule with more Gs and Cs will have the higher T
m
. This is because
the G and C bases form three hydrogen bonds between them while the A and T bases form only two hydrogen
bonds between them; more heat is required to break three hydrogen bonds than is required to break two hydrogen
bonds. The annealing temperature used for a particular primer/template combination is usually optimal at plus or
minus 7C of the primer T
m
. An annealing temperature is chosen that will give the highest amount of specific
product (one bright band on an agarose gel) with the least amount of nonspecific amplification (multiple bands on
an agarose gel). The goal is to choose a temperature at which each primer anneals to only one site on the
template. However, even at the most optimal annealing temperature, there is probably always some small
percentage of primer that will anneal to secondary sites on the template; sites that are not perfectly complementary
to the primers.

Primer Optimization: Interpreting Results

On an agarose gel, an optimized PCR amplification should give one single, bright band of the desired size. A poorly
optimized reaction will show the desired band in reduced intensity and the presence of non-specific bands of
different sizes. The AChE primers used in this experiment will produce a 553 bp PCR product.

Questions:

a) Did you obtain the 553 bp band expected for this experiment?


b) Which primer concentration seems to be optimal for amplification of this bovine acetylcholinesterase gene
segment?


c) What do you notice in the lanes with higher primer concentration? Why?


d) What do you notice in the lanes with lower primer concentration? Why?


e) What advantage does using a master mix provide the experimenter?
PCR Optimization
Student Guide
Fall 2012
33

Life Technologies & Applied Biosystems / BABEC Educational PCR Kits









For Research Use Only. Not for use in diagnostic procedures.

NOTICE TO PURCHASER: LIMITED LICENSE
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