BIOL2103

Biological Sciences Laboratory Course

Practical 2
Part A: Laboratory manual
Mapping DNA using Restriction Enzymes and
electrophoresis
Introduction:
Current research in molecular biology relies heavily on the in vitro application of
nucleic acid enzymology. In particular, the type II restriction endonucleases are
indispensable. These enzymes have the ability to cleave within double-stranded DNA
molecules at specific sequences. The particular recognition sequences are typically four
to six nucleotides in length with a two-fold axis of symmetry. The enzyme EcoRI
recognizes the hexanucleotide sequence 5’GAATTC3’ / 3’CTTAAG5’. Note that, reading
3’ to 5’, the sequence on both strands is identical (palindromic). Like many restriction
enzymes, EcoRI makes a staggered cut yielding DNA fragments with protruding,
cohesive 5’ ends. Any DNA molecule containing these ‘sticky ends’ can base-pair and
be ligated with any other to form novel recombinant molecules.

DNA fragments produced by digestion with restriction endonucleases can be

conveniently separated by agarose gel electrophoresis. Mobility of the fragments in
agarose is inversely proportional to the log of their molecular weight. Fragments can be
located in the gel by an intercalating nucleic acid stain, named Gel Red, under UV
illumination.

Analysis of fragments produced by single and multiple digestions allows one to

produce a type of genetic map showing the relative location of cleavage sites. Such a
genetic map of lambda phage DNA is shown in Figure 1. In this practical, you are going
to electrophoretically characterize single and/or double endonuclease digestions of
lambda DNA made with unknown enzymes. Comparison of the resulting fragments with
the known restriction map should allow identification of the enzymes used.

Workflow
Time Task Work done by
1:30 pm (i) Briefing (Part A)
(ii) Preparation of RE reaction
(iii) Preparation of agarose gel
2:30 pm Load sample and agarose gel electrophoresis
Students
3:00 pm (i) Briefing (Part B)
(ii) Solution preparation
4:30 pm Photo-taking

To get a brief idea, how the experiment will be performed. Please visit the
following virtual lab website before attending the practical session.
http://www.scq.ubc.ca/files/VirtualLabDNA/vlabFrame.html
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Restriction enzyme (RE) digestion
Each subgroup (2-3 students) should have a set of four 0.2ml PCR tube containing 1-2µl
RE labeled A - D. Tube A is for construction of standard curve in which lambda DNA is
digested by a restriction enzyme called HindIII. The objective of this experiment is to
identify enzymes B, C and D with the help of this standard curve.

Hint: One of the enzymes in double digestion reaction (tube D) is same as the one
in tube C

Set up single and double digestion of lambda DNA as shown below.

Single digestion (tube A-C): Double digestion (tube D):

λDNA 1.25µl λDNA 1.25µl
10X Re buffer 1.0µl 10X Re buffer 1.0µl
H 2O 6.75µl H 2O 5.75µl
RE (already in the tube) 1.0µl RE1 (already in the tube) 1.0µl
RE2 (already in the tube) 1.0µl

Incubate the tube at 37oC water bath for 15min

Agarose gel electrophoresis

(i) Preparation of an agarose gel
1. Assembly a gel tray as instructed by your demonstrator.

2. Place the comb near one end of the tray (approximately 1.5cm) and pour melted
agarose into the tray. In this practical, a 0.8% agarose gel will be prepared. (e.g. 0.8g of
agarose in 100ml of 1X TAE buffer)

3. Add 0.8g of agarose into 100ml of 1X TAE and microwave until agarose melts.

4. Add 10µl 10,000X Gel Red solution to the warm, molten agarose.

5. Pour the molten agarose to the gel tray. Do not move or handle the gel tray until the
gel solidifies (about 20min or until it appears cloudy).
6. While waiting for the solidification of the gel, prepare the DNA samples for loading by
adding tracking/loading dye. Tracking dye solution usually contains one or two dyes and
glycerol. The dye allows you to visually monitor the electrophoresis run. Glycerol is to
increase the density of the sample, so it can facilitate the loading of samples into sample
wells of the gel. Add 2µl of tracking/loading dye to each tube and mix by tapping the tube
lightly. Place the tubes in microcentrifuge and pulse spin for a few seconds. Remember
to balance the centrifuge by spacing tubes evenly.

(ii) Separation by Electrophoresis and photo taking

1. Gently remove the comb by lifting it straight up.

2. Place the tray with gel into electrophoresis tank. Be sure to place the gel with sample
wells are closest to the negative (black) electrode.

3. Fill electrophoresis tank with 1X TAE buffer to just submerge the gel.

4. Load DNA sample (12µl in total) by gently inserting pipette tip into appropriate well.
Press pipette control button slowly and steadily with your thumb to eject the sample.
Keep the pipette control button be pressed when withdrawing the pipette tip.
Loading sequence:
Lane 1: Tube A
Lane 2: Tube B
Lane 3: Tube C
Lane 4: Tube D

5. When all samples are loaded, plug the color-coded wires into the power supply
(remember black to black and red to red) and set the voltage (90V). Voltage higher than
90V may result in uneven migration of DNA. When the blue tracking dye has migrated
along the gel (about 1.5h), turn off the power supply.

6. Place the gel onto an UV illuminator and take image (follow your demonstrator’s
instruction).
Data analysis
In a given condition, the distance migrated by DNA fragments with similar conformations
on a gel is proportional to their size. The size of DNA is always expressed in kilobase
(kb).
1. Record the distances moved by all the bands on the gel photo. Measure from the
leading edge of each well to the mid point of each band.
2. Use a semi-log graph paper to present the data. The lane containing the HindIII
digest of DNA can be used as a series of reference size standards to construct a
standard curve of mobility (mm) (x-axis) vs size (kb) (y-axis) in the semi-log graph
paper.
3. By drawing the standard straight line graph, you’ll be able to estimate the size of all
unknown DNA fragments in lanes 2-4 (tube B, C and D). Knowledge of the number
and size of DNA fragments in each lane (unknown RE samples, lanes 2-4) should
allow you to identify the unknown restriction enzyme(s) in each sample.

Report
Write a concise report (including a semi-log graph paper) describing your analysis. The
report should include a copy of the gel photo, the standard curve in the semi-log graph
paper, and a table showing the number and estimated size of the DNA fragments in each
digestion and describe your interpretation leading to the identification of the enzyme(s)
responsible for the unknown digestions.
Part B: Basic techniques Solution preparation
(Adopted and modified from Bates College)

How to make simple solution and dilutions

1. Simple Dilution (Dilution Factor Method based on ratios)

A simple dilution is one in which a unit volume of a liquid material of interest is combined
with an appropriate volume of a solvent liquid to achieve the desired concentration. The
dilution factor is the total number of unit volumes in which your material will be dissolved.
The diluted material must then be thoroughly mixed to achieve the true dilution. For
example, a 1:5 dilution (verbalize as "1 to 5" dilution) entails combining 1 unit volume of
solute (the material to be diluted) + 4 unit volumes of the solvent medium (hence, 1 + 4 =
5 = dilution factor). The dilution factor is frequently expressed using exponents: 1:5
would be 5e-1; 1:100 would be 10e-2, and so on.

Example 1: Frozen orange juice concentrate is usually diluted with 4 additional cans of
cold water (the dilution solvent) giving a dilution factor of 5, i.e., the orange concentrate
represents one unit volume to which you have added 4 more cans (same unit volumes)
of water. So the orange concentrate is now distributed through 5 unit volumes. This
would be called a 1:5 dilution, and the OJ is now 1/5 as concentrated as it was originally.
So, in a simple dilution, add one less unit volume of solvent than the desired dilution
factor value.

Example 2: Suppose you must prepare 400ml of a disinfectant that requires 1:8 dilution
from a concentrated stock solution with water. Divide the volume needed by the dilution
factor (400ml / 8 = 50ml) to determine the unit volume. The dilution is then done as 50ml
concentrated disinfectant + 350ml water.

2. Serial dilution
A serial dilution is simply a series of simple dilutions which amplifies the dilution factor
quickly beginning with a small initial quantity of material (i.e., bacterial culture, a
chemical, orange juice, etc.). The source of dilution material for each step comes from
the diluted material of the previous. In a serial dilution the total dilution factor at any point
is the product of the individual dilution factors in each step up to it.

Final dilution factor (DF) = DF1 * DF2 * DF3 etc.

Example: In a typical microbiology exercise the students perform a three step 1:100
serial dilution of a bacterial culture (see figure below) in the process of quantifying the
number of viable bacteria in a culture (see figure below). Each step in this example uses
a 1ml total volume. The initial step combines 1 unit volume of bacterial culture (10µl) with
99 unit volumes of broth (990µl) = 1:100 dilution. In the second step, one unit volume of
the 1:100 dilution is combined with 99 unit volumes of broth now yielding a total dilution
of 1:100x100 = 1:10,000 dilution. Repeated again (the third step) the total dilution would
be 1:100x10,000 = 1:1,000,000 total dilution. The concentration of bacteria is now one
million times less than in the original sample.
3. Making fixed volumes of specific concentrations from liquid reagents:

V1M1=V2M2 Method

Very often you will need to make a specific volume of known concentration from stock
solutions, or perhaps due to limited availability of liquid materials (some chemicals are
very expensive and are only sold and used in small quantities, e.g., micrograms), or to
limit the amount of chemical waste. The formula below is a quick approach to calculating
such dilutions where:

V = volume, M = concentration; in whatever units you are working.

(stock solution attributes) V1M1=V2M2 (new solution attributes)

Example: Suppose you have 3ml of a stock solution of 100mg/ml ampicillin (= M1) and
you want to make 200µl (= V2) of solution having 25mg/ ml (= M2). You need to know
what volume (V1) of the stock to use as part of the 200µl total volume needed.

V1 = the volume of stock you’ll start with. This is your unknown.

M1 = 100 mg/ ml in the stock solution
V2 = total volume needed at the new concentration = 200µl = 0.2ml
M2 = the new concentration = 25 mg/ml

By algebraic rearrangement:

V1 = (V2 x M2) / M1
 V1 = (0.2ml x 25mg/ml) / 100mg/ml

and after cancelling the units,

V1 = 0.05ml, or 50µl

So, you would take 0.05ml = 50µl of stock solution and dilute it with 150µl of solvent to
get the 200µl of 25mg/ml solution needed. (Remember that the amount of solvent used
is based upon the final volume needed, so you have to subtract the starting volume form
the final to calculate it.)

4. Moles and Molar solutions (unit = M =moles/L)

Sometimes it may be more efficient to use molarity when calculating concentrations. A
mole is defined as one gram molecular weight of an element or compound, and
comprised of exactly 6.023 x 1023 atoms or molecules (this is called Avagadro's
number). The mole is therefore a unit expressing the amount of a chemical. The mass
(g) of one mole of an element is called its molecular weight (MW). When working with
compounds, the mass of one mole of the compound is called the formula weight (FW).
The distinction between MW and FW is not always simple, however, and the terms are
routinely used interchangeably in practice. Formula (or molecular) weight is always given
as part of the information on the label of a chemical bottle.

The number of moles in an arbitrary mass of a dry reagent can be calculated as:

# of moles = weight (g)/ molecular weight (g)

Molarity is the unit used to describe the number of moles of a chemical or compounds in
one liter (L) of solution and is thus a unit of concentration. By this definition, a 1.0 Molar
(1.0M) solution is equivalent to one formula weight (FW = g/mole) of a compound
dissolved in 1 liter (1.0L) of solvent (usually water).

Example 1: To prepare a liter of a simple molar solution from a dry reagent

Multiply the formula weight (or MW) by the desired molarity to determine how many
grams of reagent to use:

Chemical FW = 194.3 g/mole; to make 0.15M solution use

194.3 g/mole * 0.15 moles/L = 29.145 g/L

Example 2: To prepare a specific volume of a specific molar solution from a dry

reagent
A chemical has a FW of 180g/mole and you need 25ml (0.025L) of 0.15M (M = moles/L)
solution. How many grams of the chemical must be dissolved in 25ml water to make this
solution?
#grams/desired volume (L) = desired molarity (mole/L) * FW (g/mole)
#grams = desired volume (L) * desired molarity (mole/L) * FW (g/mole)
#grams = 0.025L * 0.15mole/L * 180g/mole after cancelling the units,
#grams = 0.675g
So, you need 0.675g/25ml
5. Concentrated stock solutions - using "X" units
Stock solutions of stable compounds are routinely maintained in labs as more
concentrated solutions that can be diluted to working strength when used in typical
applications. The usual working concentration is denoted as 1x. A solution 20 times
more concentrated would be denoted as 20x and would require a 1:20 dilution to restore
the typical working concentration.

Example: A 1x solution of a compound has a molar concentration of 0.05M for its typical
use in a lab procedure. A 20x stock would be prepared at a concentration of 20*0.05M =
1.0M. A 30x stock would be 30*0.05M = 1.5M.

Exercise

Prepare 500ml of PBS-Tris solution (pH7.4)

PBS-Tris solution (pH7.4)
10mM Tris (FW:121.1)
0.15M NaCl (FW: 58.44)
10mM NaH2PO4 (FW: 119.98)

Calculate the amount of chemicals required to prepare this

solution and show your calculation to the demonstrator.
He/she will then show you how to use the balance and prepare
the solution.
(A) Weighting
Types of balances
(i) Top-loading balance. It weighs to an accuracy of ± 1mg and are suitable for most
weighings of amounts that are specified to only two or three significant figures.

(ii) Analytical balance. It weighs to an accuracy of ± 0.1mg and must be used

whenever you desire four or more significant figure accuracy.

Caution

More…..
- Don’t handle objects to be weighed with bare hands.
- Don’t leave anything exceeding the weighing capacity on the pan.
- Don’t spill chemicals inside the balance enclosure. If a spill occurs, clean it up.
- To be weighed accurately, all objects must be at room temperature. A warm object sets
up convection currents inside the balance enclosure, which will make an object appear
lighter than it really is. Also, warm air inside the enclosure is less dense than the air that
it displaces and this also leads to a negative determinate error.
- Don’t weigh chemicals directly in contact with the balance pan. Use containers such as
beakers, flasks and weighing bottles.
Name:___________________________ University No.:____________________________

You have the following chemicals and stock solutions in your laboratory:
Tris-base powder (FW: 121.4) 0.5M EDTA
NaCl (FW: 58.44) NP-40
200 mM PMSF Glycerol

Calculate the amount of chemicals and stock solutions required to prepare 200ml of IP lysis
buffer containing 50mM Tris-base, 0.15M NaCl, 2mM PMSF, 2mM EDTA, 1% NP-40 and
10% glycerol. Show your calculation.
 Three Cycle Semi-Log
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Distance (cm)