LAB MANUAL 2022

BMS531 & BMS537

METHODS IN MOLECULAR BIOLOGY I
METHODS IN RECOMBINANT DNA
TECHNOLOGY

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Introduction

Molecular Biology methods are not unlike cooking. Get a good recipe, collect all the
ingredients, mix things together at the appropriate time, bake, and done. Yet there are good
cooks and bad cooks. Sometimes the difference can be very subtle, and practice make perfect.
The basic rules applies :

Understand the recipe

Use the correct ingredients
Add the correct amounts
Follow recipe strictly (order, timing etc).
Keep very tidy and clean.
Ask if you are not sure!
Be consistent

The one big different is that reagents for molecular biology can be very expensive.

Please take note of the following safety precautions :

To protect yourself :

1. Wear protective clothing: laboratory coat, safety glasses and enclosed footware.
The use of gloves is somewhat debatable, but is advisable.
2. Be aware that some chemicals used are toxic. Take special care. Always find out the
hazard nature of a chemical before you work with it. If unknown, always assume it is
toxic. Use fume cupboards, laminar flows or biohazard chambers when in doubt.
e.g. phenol, chloroform - corrosive, burns
ethidium bromide - mutagen
3. Dispose of waste properly.
4. Some equipment are potentially dangerously.
e.g. Electrophoresis kit have high voltages,
Centrifuges can literally fly off the bench if grossly unbalanced.
UV light is bad for eyes
If unsure, always ask.
5. Learn proper microbiology techniques. Although most of the time the bacteria we
use are generally regarded as harmless.
6. Good laboratory practice always pays. Keep things neat and tidy.

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To protect the experiments :

1. Your hands are full of dead cells which are choke-ful of DNase, RNase and proteases.
These enzymes are stable and remain active for sometime, and can really mess up
your samples. Handle things very carefully. Wear gloves if necessary (compulsory
when handling RNA). Autoclave all containers and solutions. Remember, you are
your worse enemy.
2. Most enzymes are very expensive and unstable, so keep on ice at all times.
Immediately return an enzyme to the freezer once you are done.
3. Do not recycle pipette tips or eppndorf tubes etc. They are meant to be disposable.
4. The key to many of the techniques described in this manual is the ability to pipette
small volumes carefully and accurately. It is essential when pipetting small volumes
to ensure that the tip of the pipette is touching the surface of the container before
expelling the solution. Always use clean pipette tips to avoid cross contamination.
5. Pulse spin microfuge tubes to ensure that all of the added components of reaction
mixtures are mixed at the bottom of the tube.
6. Proper and careful labeling. All eppendorf tubes look alike, so label properly!
Imagine if you drop a rack……..

To help make things work :

1. Each student is required to bring their own

• fine tip permanent markers – of at least two different colours. You will find these
markers handy as there will be lots of labeling to do.
• masking tape, to label Schott bottles, beakers, conical flasks or other glassware
2. Almost all molecular biology reactions are enzymatic or biochemical reactions.
That means they take time and require the correct conditions to happen. Be patient
and be meticulous.
3. As reagents are very expensive, we typically use very small amounts. Pipette
carefully and be confident of what you have done.

Get use to small measurements !

1 kg = 1000 g
1g = 1000 mg
1 mg = 1000 ug (microgram)
1 ug = 1000 ng (nanogram)
1 ng = 1000 pg (picogram)

1l = 1000 ml
1 ml = 1000 ul (microliter)

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 LAB 1 & 2 : DNA EXTRACTION

Objective

To extract DNA from bacteria cells.

Introduction

Since DNA is the blueprint for life, everything living contains DNA. DNA isolation is one of
the most basic and essential techniques in the study of DNA. The extraction of DNA from cells
and its purification are of primary importance to the field of biotechnology and forensics.
Extraction and purification of DNA are the first steps in the analysis and manipulation of DNA
that allow scientists to detect genetic disorders, produce DNA fingerprints of individuals, and
even create genetically engineered organisms that can produce beneficial products such as
insulin, antibiotics, and hormones.

Many different methods and technologies are available for the isolation of genomic DNA. The
goals however, remain the same to release the nuclear DNA from the cell, and to precipitate
DNA out of solution so that it can be seen. In general, all methods involve disruption and lysis of
the starting material followed by the removal of proteins and other contaminants and finally
recovery of the DNA. Removal of proteins is typically achieved by digestion with proteinase K,
followed by salting-out, organic extraction, or binding of the DNA to a solid-phase support
(either anion-exchange or silica technology).

DNA is usually recovered by precipitation using ethanol or isopropanol. The choice of a method
depends on many factors: the required quantity and molecular weight of the DNA, the purity
and others

Materials

Micropippette P1000 & Sterile Tips (blue)

Micropippette P20 & Sterile Tips (yellow)
1.5 mL Eppendorf tubes (sterile)
Heat block
Crushed ice
Microcentrifuge
E.coli cells
B. subtilis cells

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Things to do before starting

• DNA extraction will be performed using the DNeasy Blood & Tissue Kits by
Qiagen. Hence, the protocol for the DNA extraction is as according to the
manufacturer’s instructions in the DNeasy Blood & Tissue Handbook PDF which can be
downloaded online.
• Before you start the experiment, you must first check that the kit given to you contains
all the necessasary reagents needed to carry out the experiment.

KIT CONTENTS

No DNeasy Blood & Tissue Kit (50) (250)

Number of preps 50 250
1 DNeasy Mini Spin Columns (colorless) in 2 ml
50 250
Collection Tubes
2 Collection Tubes (2 ml) 100 500
3 Buffer ATL 14ml 50ml
4 Buffer AL* 12ml 2 X 33ml
5 Buffer AW1 (concentrate)*† 19ml 98ml
6 Buffer AW2 (concentrate)†‡ 13ml 66ml
7 Buffer AE 2 X 15ml 2 X 60ml
8 Proteinase K 1.25ml 6ml

* Contains a chaotropic salt. Not compatible with disinfecting agents containing bleach
† Buffer AW1 and Buffer AW2 are supplied as concentrates. Add ethanol (96–100%) according to the bottle label
before use. Tick the the check box on the bottle label to indicate that ethanol has been added. Write the date.
‡ Contains sodium azide as a preservative

• All centrifugation steps are carried out at room temperature (15–25°C) in a

microcentrifuge.
• Buffer ATL and Buffer AL may form precipitates upon storage. If necessary, warm to 56°C
until the precipitates have fully dissolved.
• Buffer AW1 and Buffer AW2 are supplied as concentrates. Before using for the first time,
add the appropriate amount of ethanol (96–100%) as indicated on the bottle to obtain a
working solution.
• Avoid repeated thawing and freezing of samples, because this will lead to reduced DNA
size.

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 LAB 1: DNA extraction of Gram Negative bacteria

Methodology

1. Preheat a thermomixer, shaking water bath or rocking platform to 56°C for use in step 4.
2. Harvest cells (maximum 2 x 109 cells) in a microcentrifuge tube by centrifuging for 10 min
at 5000 x g (7500 rpm). Discard supernatant.
3. Resuspend pellet in 180 µl Buffer ATL.
4. Add 20 µl Proteinase K. Mix thoroughly by vortexing, and incubate at 56°C until the cells
are completely lysed. Vortex occasionally during incubation to disperse the sample or
place in a thermomixer, shaking water bath or on a rocking platform. Lysis time varies
depending on the type of cells processed. For Gram negative bacteria, incubate the cells
for at least 30 mins for the cells to lyse.
Optional: If RNA-free genomic DNA is required, add 4 µl RNase A (100 mg/ml), mix by
vortexing, and incubate for 2 min at room temperature (15–25°C) before continuing.
5. Vortex for 15 s. Add 200 µl Buffer AL to the sample, and mix thoroughly by vortexing. Then
add 200 µl ethanol (96–100%), and mix again by vortexing. It is essential that the sample,
Buffer AL, and ethanol are mixed immediately and thoroughly by vortexing or pipetting to
yield a homogeneous solution.
A white precipitate may form on addition of Buffer AL and ethanol. This precipitate does not
interfere with the DNeasy procedure. If gelatinous lysate is formed after addition of Buffer
AL and ethanol, vortex the solution or shake vigorously to homogenize the preparation.
6. Pipet the mixture from step 5 (including any precipitate) into the DNeasy Mini spin column
placed in a 2 ml collection tube (provided). Label the tubes. Centrifuge at ≥6000 x g (8000
rpm) for 1 min. Discard flow-through and the collection tube.
7. Place the DNeasy Mini spin column in a new 2 ml collection tube, add 500 µl Buffer AW1
and centrifuge for 1 min at ≥6000 x g (8000 rpm). Discard flow-through and collection tube.
8. Place the DNeasy Mini spin column in a new 2 ml collection tube, add 500 µl Buffer AW2,
and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane. Discard
flow-through and collection tube.
It is important to dry the membrane of the DNeasy Mini spin column, since residual ethanol
may interfere with subsequent reactions. This centrifugation step ensures that no residual
ethanol will be carried over during the following elution.
Following the centrifugation step, remove the DNeasy Mini spin column carefully so that
the column does not come into contact with the flow-through, since this will result in
carryover of ethanol. If carryover of ethanol occurs, empty the collection tube, then reuse
it in another centrifugation for 1 min at 20,000 x g (14,000rpm).
9. Place the DNeasy Mini spin column in a clean labelled microcentrifuge tube (not provided),
and pipet 200 µl Buffer AE directly onto the DNeasy membrane. Incubate at room
temperature for 1 min, and then centrifuge for 1 min at ≥ 6000 x g (8000 rpm) to elute.
Elution with 100 µl (instead of 200 µl) increases the final DNA concentration in the eluate,
but also decreases the overall DNA yield.
Note: Do not elute more than 200 µl into a 1.5 ml microcentrifuge tube because the DNeasy
Mini spin column will come into contact with the eluate
10. Keep the extracted DNA for Lab 3 & 4.

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Preparation and calculations of 10X TBE pH 8.0

TBE Buffer is a solution used in Agarose Gel Electrophoresis for the separation of nucleic acids
(DNA and RNA). Hence, students are required to prepare this buffer ahead for your future lab
sessions (Lab 4 & 6). However, each group will only use a small volume of 1 X TBE. Because of
that, only ONE group (voluntarily) is enough to carry the task to prepare a 1 L bottle of 10 X TBE
for the need of the whole class throughout the semester.

10X TBE pH 8.0

(Tris-Borate EDTA buffer, 10X concentrated)

108.0 g Tris-base,
55.0 g boric acid,
9.3 g EDTA

Weigh and and dissolve each of the chemicals one after another, in the exact order in
approximately 700 ml dH2O. Use a magnetic stirrer to stir until the solution gets clear
Check that pH is about 8.2 to 8.4. If not, adjust with HCl or NaOH (Careful!)
Adjust volume to 1000 ml in a measuring cylinder.
Autoclave at 121°C for 15minutes .

When the buffer has cool down, dilute to 500 ml 1 X TBE in a Schott bottle. Label the bottle
according to your group using a masking tape. Write the date. Keep the 1 X TBE for future lab
sessions.

You can use the exercise below to practice your calculations.

1. Calculate the amount of chemicals needed to prepare the following stock solutions

VOLUME CONCENTRATION TRIS (g) BORIC ACID EDTA

(ml) (g) (g)

1000 10X TBE 108 55 9.3

500 10X TBE

1000 5X TBE

650 5X TBE

375 10X TBE

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2. TBE is always made up of a 10X concentrated stock solution so that it can be
stored conveniently. Before using, it is diluted to the working concentration of 1X.
Calculate the amount of TBE stock needed to prepare the following solution.

VOLUME VOLUME OF FINAL

STOCK WORKING OF dH2O VOLUME
SOLUTION SOLUTION STOCK (ml) (ml)
(ml)

10X TBE 1X TBE 1000

10X TBE 1X TBE 500

25X TBE 1X TBE 1000

25X TBE 1X TBE 750

40X TBE 1X TBE 500

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 LAB 2: DNA extraction of Gram Positive bacteria

Things to do before starting

Enzymatic lysis buffer is necessary for pretreatment of Gram-positive bacteria. The recipe for
the enzymatic lysis buffer is as follows:
➢ 20 mM Tris-Cl, pH 8.0
➢ 2 mM sodium EDTA
➢ 1.2% Triton® X-100
➢ Immediately before use, add lysozyme to 20 mg/ml
(Practice your calculations: If you are given stock solutions of 1 M 1 M Tris-HCl,0.5 M EDTA
and 100% Triton, calculate how much of each of the stock solution is needed to prepare a 250ml
solution of enzymatic lysis buffer).

In this experiment, the enzymatic lysis buffer has been prepared for you.

Methodology

1. Preheat a a heating block or water bath to 56°C for use in step 5.

2. Harvest cells (maximum 2 x 109 cells) in a microcentrifuge tube by centrifuging for 10 min
at 5000 x g (7500 rpm). Discard supernatant.
3. Resuspend bacterial pellet in 180 µl enzymatic lysis buffer. Incubate for at least 30 min at
37°C.
4. Add 25 µl Proteinase K and 200 µl Buffer AL. Mix by vortexing. Note: Do not add Proteinase
K directly to Buffer AL. Ensure that ethanol has not been added to Buffer AL.
5. Incubate at 56°C for 30 min. Optional: If required, incubate at 95°C for 15 min to inactivate
pathogens. Note that incubation at 95°C can lead to some DNA degradation.
6. Add 200 µl ethanol (96–100%) to the sample, and mix thoroughly by vortexing. It is
important that the sample and the ethanol are mixed thoroughly to yield a homogeneous
solution. A white precipitate may form on addition of ethanol. This precipitate does not
interfere with the DNeasy procedure.
7. Continue with step 6 of the protocol “DNA extraction from Gram Negative bacteria” on
page 5.

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 LAB 3 : POLYMERASE CHAIN REACTION (PCR)

Objectives

To use PCR to amplify a DNA fragment

Materials

P1000 & Sterile Tips (blue) P100 & Sterile Tips (yellow)
1.5 mL Eppendorf tubes (sterile) P10 & Sterile Tips (white)
Sterile saline PCR reagents (Promega PCR kit)
Micro-centrifuge crushed ice
Bacterial DNA from Lab 1 & 2

The following are the ‘ingredients’ for PCR using Promega PCR kit :
• 5x buffer
There are two types of buffers given. One is colourless and the other comes with dye
incorporated hence they are green in colour.
For this lab, choose the green buffer as you can load them straight to the loading wells of
agarose gel later after PCR is completed without having to add the loading dye to view the
bands.

• 25 mM Magnesium chloride

• Primers 10 µM
We need the Forward and Backward primers to amplify the DNA region of interest.
The dried primers are made to a 100 µM concentration stock solution by adding appropriate
amount of 1M Tris-EDTA buffer pH 8.0 . A working solution of 10 M is obtained by a 1:10
dilution of the stock solution using sterile water.

• dNTP's stock solutions 10 mM

A working solutions of dNTP’s, with a concentration of 10 mM of each of the dNTP’s is made
up from 100 mM stock solutions of dATP, dTTP, dGTP, and dCTP.

• Thermostable Taq polymerase

DNA polymerase enzyme from the bacteria Thermus aquaticus

• DNA sample with the target gene

The DNA samples that you are going to use in this experiment will be the genomic DNA
extracted in Lab 1 & 2

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Introduction

In this experiment, you are going to use the polymerase chain reaction technique to amplify a
16S RNA gene fragment using a pair of primers as follows.

27F: AGA GTT TGA TCM TGG CTC AG

1492R: CGG TTA CCT TGT TAC GAC TT

The 16S rRNA gene is used as the standard for classification and identification of microbes,
because it is present in most microbes. For classification, the 16S rRNA gene is used for
phylogenetic studies as it is highly conserved between different species of bacteria and
archaea. In addition to highly conserved primer binding sites, 16S rRNA gene sequences also
contain hypervariable regions that can provide species-specific signature sequences useful for
identification of bacteria. As a result, 16S rRNA gene sequencing has become prevalent in
medical microbiology as a rapid and cheap alternative to phenotypic methods of bacterial
identification. Although it was originally used to identify bacteria, 16S sequencing was
subsequently found to be capable of reclassifying bacteria into completely new species or even
genera.It has also been used to describe new species that have never been successfully
cultured.

Thoughts to ponder
• List down the advantages/disadvantages of using traditional biochemical methods in
determining bacterial ID
• What about using automated system for bacterial ID? What are the pros and cons?
• Base on the primer sequence given above, determine the size of your expected
amplicon.

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Methodology

Add the following contents in an eppendorf tube except for the DNA sample. Keep in ice
(WHY??).
Volume per X
Volume per
Reaction
Component* Concentration Final conc. Reaction
(Master mix)
(µl)
(µl)
Sterile dH2O
5x buffer 5X 1X 10
MgCl2 25 mM 1.0 – 4 mM
dNTP’s 10 mM 0.2 mM
Primer (Forward) 10 µm 0.1 – 1.0 µm
Primer (Reverse) 10 µm 0.1 – 1.0 µm
Taq polymerase 5U/µl 1.25U
DNA Sample* 5
TOTAL 50

* do not add until everybody’s ready

2. Mix by vortex, followed by a brief (~10 sec) pulse in the centrifuge. Label clearly.

3. Once everybody has prepared the PCR mix, add 5 ul of the crude DNA extract (from Lab 1 &
2) and immediately place in the thermocycler block.

4. Run the following cycling program

PCR conditions

Temp Time Cycle x

Initial denaturation 94oC 10 min 1

Denaturation 94oC 30 s
Annealing 55oC 15 s 35
Extension 72oC 1 min

Final cycle 72oC 10 min 1

Hold 4o C forever

5. After completion, remove your reactions tubes. Keep the labelled PCR products to be
viewed in Lab 4.

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 LAB 4 : Agarose Gel Electrophoresis

Objectives

To separate DNA fragments (from Lab 1 & 2) on an agarose gel

To examine the PCR products (from Lab 3) using gel electrophoresis.
To estimate the size of the DNA by comparison with a known DNA ladder

Introduction
(modified from SBB321 Molecular Biology Techniques (Practical Manual U.Deakin)

The standard method used to separate, identify and purify DNA fragments is electrophoresis
through agarose gels. Samples are applied to the gel with the aid of a gel loading buffer
containing glycerol which helps the sample to sediment into the wells. Two tracking dyes will
be used (i.e. bromophenol blue and xylene cyanol).

A direct current is applied to the gel and the DNA migrates through the gel at a rate dependent
upon: (i) the molecular size of the particle, (ii) the agarose concentration, (iii) the conformation
of the DNA (i.e. superhelical compared with linear), (iv) the current applied to the gel.

Bands of DNA in the gel will be located by staining with a low concentration of the fluorescent,
intercalating dye, ethidium bromide. As little as 1 ng of dsDNA can be detected by direct
examination of the gel in UV light using excitation wavelengths of 260, 300 or 360nm. However,
Ethidium bromide is a powerful mutagen so caution must be taken whilst handling gels or
electrophoresis solutions and tanks.

GelRed is another intercalating nucleic acid stain used in molecular biology for agarose gel
electrophoresis. GelRed is structurally closely related to ethidium bromide .Its fluorophore,
and therefore its optical properties, are essentially identical to those of ethidium bromide.
When exposed to ultraviolet light, it will fluoresce with an orange color that strongly intensifies
after binding to DNA. The substance is marketed as a less toxic and more sensitive alternative
to ethidium bromide

In our laboratory, we’ll be using GelRed for DNA staining. Once localized, the band size may
be determined by comparison with a molecular weight marker (in our case 1 kb plus DNA
ladder). Specific DNA bands may then be recovered from the gel for further study.

Thoughts to ponder

What are the advantages and disadvantages of using EtBR and GelRed as NA dye in gel
electrophoresis.

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Range of Separation for Agarose Gels

Agarose Optimal Range of Separation

% kb
0.3 60 - 5.0
0.6 20 - 1.0
0.7 10 - 0.8
0.9 7 - 0.5
1.2 6 - 0.4
1.5 4 - 0.2
2.0 3 - 0.1

Materials

Micropipette and tips

Agarose
10 X TBE
GelRed (check the concentration)
DNA ladder
6XLoading buffer (10mM Tris-HCl pH 7.6, 0.03% bromophenol blue, 0.03% xylene cyanol FF,
60mM EDTA, 60% glycerol )
Mini horizontal gel unit & Power pack

Methodology
1. Dilute the 10X TBE buffer given to a concentration of 1X. Each group will need 500 ml.
2. Weigh out 1.0 g of agarose. Suspend in 100 ml TBE in a 250ml Schott Bottle. Label the
bottle as 1.0% agarose gel using a masking tape. Write down your group and the date.
3. Boil in a microwave to dissolve the agar. Cover with cling film to prevent excessive
evaporation. The agar must be completely dissolved. No lumps or floaties.
4. Let cool on the bench until ~ 45oC.
5. Add ….. ul of the Gel Red solution.
6. Pour the agar slurry into the casting tray. Immediately put in place the combs for
making wells.
7. Leave the remaining 1.0% agarose gel in the Schott Bottle for future use.
8. Let the gel harden. Then place into the gel unit in the correct orientation.
9. Pour the TBE into the gel unit until the gel is just submerged.
10. Load the 1kb DNA ladder into the leftmost well.
11. Mix 5 ul of the DNA samples with 2 ul of loading dye and 5 ul of TE. Carefully load into
the individual wells. (How do you calculate this??).
12. Close the cover, connect the electrodes and run the gel at 90V for 60 minutes. If time
permits, run the gel at 80V for 80 minutes for a better separation.

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Observation

1. After completion, carefully remove the gel from the unit.

2. Place on the uv transillumintor.
3. Observe the DNA bands on the gel. The gel red intercalates with the DNA molecules and
produce a bright orange fluorescent when excited by uv light.
Wear protective glasses or face shields
4. Take a picture with the camera.
5. Estimate the size of the DNA band by comparison with the known sizes of the DNA
ladder. Plot the log molecular weight of each band of the DNA ladder against the
distance it has travelled from the wells (cm). Use this as a standard curve to estimate the
sizes of other bands.

sample well

Distance travelled
by this particular
band.

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 LAB 5: Plasmid extraction

Objectives

To extract pUC19 plasmid DNA from bacteria cells.

Materials
P1000 & Sterile Tips (blue)
P20 & Sterile Tips (yellow)
1.5 mL Eppendorf tubes (sterile)
Plasmid isolation kit (the protocol differs depending on the kit used)
Microcentrifuge

NOTE: THERE ARE TWO METHODS OF PLASMID EXTRACTION GIVEN IN THIS

MANUAL, CHOOSE ONLY ONE, DEPENDING ON THE KIT AVAILABLE IN THE LAB

1. PLASMID KIT – Plasmid DNA Extraction/Purification using the QIAprep Spin

Miniprep Kit by QIAGEN

Methodology

Note: All protocol steps should be carried out at room

temperature

A Preparation of cell lysate

1. You will be given an E.coli culture that
contains pUC19 plasmid, grown for 24 hr at
37oC, 200 rpm.
2. Pipette 1.0 ml of this culture into an eppendorf
tube.
3. Collect the cells by centrifuging in a microfuge
at 13000 rpm for 5 minutes. Remove the
supernatant.
Cell resuspension solution contains EDTA which makes
4. Follow instruction given by the manufacturer the bacterial outer membrane permeable and RNase
of the kit : which degrades RNA.
The NaOH denatures the proteins and disrupts the
5. Add 250μl of Cell Resuspension Buffer (P1) and chromosome. The detergent, SDS, lyses the bacteria
resuspend the cell pellet by brief vortexing. allowing the plasmids to leak out of the cells.
The bacteria should be resuspended
completely by vortexing or pipetting up and
down until no cell clumps remain. The SDS and potassium acetate forms a precipitate
with the proteins and chromosomal DNA while the
6. Add 250μl of Cell Lysis Solution (Buffer P2) and plasmid remains in solution. The acetic acid neutralises
mix thoroughly by inverting the tube four the alkali used in the previous step
times (do not vortex as this will result in The plasmid (in solution) is separated from the
shearing of genomic DNA and contamination precipitated proteins, chromosome and cell debris.
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 of plasmid). Continue inverting the tube until
the solution becomes viscous and slightly
clear. Do not allow the lysis reaction to
proceed for more than 5 min.
7. Add 350μl of Neutralisation Solution (N3) and
immediately mix by inverting the tube several
times, gently. A white precipitate should form.
8. Spin at full speed in the microcentrifuge for 10
min to pellet the precipitate. A compact white
pellet will form. Assemble a spin colum while
you are waiting.
B Purification of plasmid DNA by matrix binding
9. Carefully apply the clear supernatant
(approximately 800 µl) into the QIAprep Spin Under high salt conditions Nucleic acids binds (plasmid
column by pipetting. Do not include any DNA) to Spin Column.
white material.
10. Insert the column into a collection tube and
spin at max speed for 1 minute.
11. Discard the flowthrough and reinsert the Contaminating proteins and salts are washed away. The
washing solution contains salt ethanol and a small amount
column.
of salt so that mono-nucleotides are removed but plasmid
12. Wash the spin column by adding 500 ul of DNA remains bound.
Buffer PB and spin again for 1 minute. Discard Remaining wash solution is removed.
the flowthrough.
13. Wash the spin column again by adding 750 ul
of Buffer PE and spin for 1 minute. Discard the
flowthrough.
14. Spin again for another 1 minute to remove all
all the residual wash buffer.

C Elution of plasmid DNA from matrix

15. Transfer the QIAprep Spin column to a new
sterile eppendorf tube.
16. To elute the plasmid, add 50 μl Buffer EB (10
mM Tris·Cl, pH 8.5) or water to the center of Water causes the DNA to be released from the resin. The
each of the QIAprep 2.0 Spin Column. plasmid DNA solution is then collected in a fresh eppendoff
when the column is spun in the microcentrifuge.
17. Incubate for 1 min
18. Spin for 1 min.
19. Discard the column, and keep the plasmid
DNA and label for the next lab session. Store
at -20oC.
20. (For long term storage, elute in TE instead of
water).

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2. PLASMID KIT – Plasmid DNA Extraction/Purification using NucleoSpin ® Plasmid kit

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 LAB 6 : Restriction Enzyme digest of DNA

Objectives

To cut the pUC19 plasmid (circular DNA) islolated in Experiment 5 with restriction enzymes

Restriction enzymes are enzymes that cut DNA in a sequence speicifc manner. For example, the
enzyme EcoRI make a cut when it sees the sequence -GAATTC- on a stretch of DNA. This makes
REs very useful when you want to cut and join different pieces of DNA.

We will cut the plasmid pUC19 (which you extracted in LAB 5) with several enzymes and analyse
the restriction patterns. We will also cut the lambda phage DNA which is a linear DNA as a
comparison.

Materials

The usual pipettes, tips and tubes.

Restriction Enzymes – EcoR1 and BamH1
Respective Buffers
pUC19 Plasmid DNA that you isolated in Lab 5
Lambda DNA (linear DNA) for comparison
pUC19 commercial plasmid

Methodology

(Instructor : Explain the use of buffers, enzyme units and demonstrate preparation of
mastermix)

1. For Group 1, prepare the following reaction in a sterile 0.5 ml tube:

10X RE Buffer 2.0 ul

Plasmid DNA* 5.0 ul
Enzyme 0.1 ul
_______
Water to 20.0 ul

* Use the commercial pUC19 DNA (pUC19C )

2. Prepare samples for both single and double digestion.
3. For Group 2 prepare a similar set of reaction using the linear λ DNA instead for
comparison
4. Mix well and spin briefly to collect everything at the bottom.
5. Incubate at 37oC for at least 1 hour.

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6. When the incubation is proceeding, prepare an agarose gel as in LAB 4.
7. At the end of the 1 hour incubation period, remove the tube and heat at 60 oC for 20
minutes to inactivate the enzyme.
8. Spin down the content of the reaction tube.
9. Load a 5 ul sample onto the agarose gel. Also include a sample of the uncut DNA of the
pUC19 and lambda DNA for comparison. Don’t forget the DNA ladder. Use the table
below to help you determine what to load into the wells of your gel.
10. Electrophorese the samples at 90V for 1 hour.
11. Then observe under uv and obtain an image of the gel.
12. Use the imager’s software to automatically calculate the size of each fragment.
13. When doing lab report, include both gel pictures of both groups and compare the
results.

Well Group 1 Group 2

1 DNA ladder 100bp DNA ladder 100bp

2 pUC19 uncut (that you isolate pUC19 uncut (that you isolate in
in LAB 5) LAB 5)
3 pUC19C uncut (commercial) λ DNA uncut

4 pUC19C + EcoR1 λ DNA + EcoR1

5 pUC19C + BamH1 λ DNA + BamH1

6 pUC19C + EcoR1 + BamH1 λ DNA + EcoR1 + BamH1

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 LAB 7 : Transformation of Bacteria

Objective

To introduce a plasmid vector DNA into bacteria cells.

Materials
P1000 & Sterile Tips (blue)
P20 & Sterile Tips (yellow)
1.5 mL Eppendorf tubes (sterile)
Ice bucket
Waterbath at 42oC
Microcentrifuge
E.coli DH5α cells
pUC19 plasmid DNA (10 ng/ul)

LB broth (per liter : 10g tryptone, 10g yeast extract, 10g NaCl)

Transformation solution (TSS ) (0.5ml):

LB broth with 10% (wt/vol) Polyethylene glycol 3350
5% (vol/vol) Dimethyl sulfoxide
50 mM MgCl2 (or MgSO4)

LB broth (10 ml)

LB Agar + 100 mg/l ampicillin (10 plates)
LB Agar plates ( 2 plates)

In DNA cloning, a foreign piece of DNA fragment is ligated to a vector to form a recombinant
DNA molecule. The function of the vector is to carry the foreign DNA into a host cell, maintain
the foreign DNA so that it is not lost, and propagate the DNA as the host cell divides. The
simplest DNA cloning vector is a plasmid, a circular piece of DNA that can be found naturally in
some bacteria. Plasmids have short sequences of DNA called origin of replication which allows
them to maintain themselves and replicate with the host cell’s DNA.

In this practical we will learn how to introduce (‘transform’) a plasmid vector into a bacterial
cell. The plasmid we use is called pUC19 and the host cell is a special strain of E.coli known as
DH5α. Both are routinely used in DNA cloning experiments. In order for the plasmid to be taken
up, the E.coli DH5 cells must be first induced into a competent state. Non-competent cells take
up very little DNA and are not useful. Competent cells are usually made by treating them with
cold CaCl2. This is however a lengthy and tedious step, and furthermore cells made competent
in this way must always be kept at very low temperature (-80oC) or the will lose their comptency.
We will use a one-step procedure employing polyethylene glycol (PEG) instead, which is much
easier. The PEG-competent cells we made, however will not be as good as CaCl 2- competent
cells, but can be stored in a normal fridge.

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A Transformation of E.coli cells

(The first three steps has been done for you by the lab technician)
1. Inoculate a fresh overnight culture of E.coli DH5α into LB broth at 1:100 dilution.
2. Incubate at 37oC, 250 rpm for 3-4 hours until an OD600 of 0.3 – 0.4.
3. Thaw a tube of 500 ul TSS and keep cold on ice.
4. Pipette 100 ul of the E.coli DH5α cells into the ice-cold TSS. Keep on ice for 2-5 minutes.
5. Add 100 ul ml of the cells into into an empty tube.
6. Pipette 1 ul of pUC19 plasmid into the tube and place on ice.
Label clearly as ‘P’ (plasmid).
7. Also add 100 ul of the E.coli cells into another empty tube.
Label this ‘C’ (control)
8. Incubate bothl tubes on ice for 30 minutes.
9. Heat shock at 420C in a water bath for 20 seconds. Place on ice for a further 2 minutes.
10. Add 0.9 ml of LB+20mM glucose to both tubes and incubate at 37oC, 225 rpm for 1 hour.
11. Plate 200 ul each of ‘P’ on five LB+Amp plates.
Label these P1 – P5
Incubate overnight at 37oC.
12. Plate 500 ul of ‘C’ on one LB+Amp plates. Do duplicate.
Label as ‘C500’. Incubate.
13. Plate 1 ul of ‘C’ on LB plates only ( no Ampicillin!). Do not forget to duplicate your
plate.
14. On the next day, count the number of colonies on all plates.
15. Use the sheet next page to do the necessasary calculation.

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7A Count the plates on which you plated the transformed bacteria (which plate?)
Plate P1 P2 P3 P4 P5 Total
Counts
(colonies)
This would give you the number of transformants/ml

7B Count your control plates (which plate?)

Plate C1 C1 Average Original cells/ml C500
(LB + Amp)
Counts
(colonies)
Use C1 to estimate the number of cells/ml in the original cell suspension

There should be no colonies on C500. Wh y? What is the purpose of this plate?

Calculate the amount of plasmid that was added into the transformation mix.

Calculate the transformation efficiency of the plasmid against the host cell -
(no. of transformants/ng plasmid)

Assuming that every colony grew on the LB Amp plate carries one copy of the pUC19 plasmid,
calculate the percentage of bacteria that has been successfully transformed.

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 LAB 8A : Analysis of DNA sequences

Objective

To analyse DNA sequences using computers

Materials
PC with Windows XP
ApE software

ApE is a small program for displaying and analysing DNA sequences. Best of all, it is available
free on the web. We will learn how to use the basic features to find out useful information
about a particular DNA sequence.
Your first take home test will require a good working knowledge of this software, so pay
attention and follow all the steps.
If you are not familiar with the Windows OS, please get a friend to help you out.

PAY ATTENTION AND DO ALL THE EXERCISES. YOU WILL NEED THIS FOR YOUR TEST.

1. Double click on the Shortcut to APE to launch the program

2. A series of icons will be available at the toolbar. Write down the function of each icon.

(a) (b) ………………………………………………………………………(r)

(a) (j)
(b) (k)
(c) (l)
(d) (m)
(e) (n)
(f) (o)
(g) (p)
(h) (q)
(i) (r)

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3. Select the Open file icon from the toolbar.
4. Select the folder APE files from the desktop. Click open.
5. Select the file lambda.str and click open. The DNA sequence for the bacteriophage
lambda is now loaded into APE.
6. Write down the total length of the lambda DNA.
7. Click on the Graphic Map icon. A map of the sequence will be display.
8. Click on the Linear button. The icon will change to

9. Click the Graphic Map icon again. Compare with what you see in step 7. Then close
both windows. Change the icon back to Linear by clicking it.

10. Move the cursor over the sequence. The number on the top left corner changes. What
does the number represents?

11. Click anywhere within the sequence. The third number on your top left corner change.
What does this number represents?
12. What are the numbers on the sides of the sequence box for?

13. Open the Enzyme Selector dialog. You can do this by either clicking the Enzyme
Selector icon or select Enzyme from the toolbar and then select Enzyme Selector or by
pressing Ctrl-E.
14. What do the names and the number in brackets represent?

15. Select ‘HindIII’. The name will now be highlighted in red. Place your cursor over the
highlighted text. Write down the recognition sequence for this enzyme.

16. Select the Graphic Map. A map of the lambda DNA and the position of HindIII
recognition sites (or restriction sites) is displayed. The numbers indicate the position of
the sites. Draw it here :

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17. Go back to the APE window. Select the Digestion Window. A figure will open up. It
shows what you would expect to see if a sample of lambda DNA digested to completion
with HindIII is ran on an agarose gel. Compare this to your results in Practical #2.
Write down what each section of the figure represent.

17. Review the picture of your gel from Practical #2 here. Place a copy of the picture here.
Write down and indicate using arrows the size of each and every band you can see. Can
you locate all seven bands? Why?

Band No: Size (bp)

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18. Repeat this exercise(except step 17) with the restriction enzyme AflII. Then repeat with a
combination of AflII and SacII.

For each exercise, write down :

a) number of restriction sites
b) position of restriction sites
c) numbers of fragments generated in the digest
d) size of each fragment
e) draw a picture of the digest (roughly, no need to draw the ladder) or use Windows
Prt-Scr function to obtain an image.

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 LAB 8B : ApE – Sequence analysis

Objective

To analyse DNA sequences using computers

Materials
PC with Windows XP
ApE software

The is the second part of the lesson on APE. I hope you have been trying out the program on
your own and is no well familiarise with its basic features. We will go on to explore other
useful features of this sequence analysis program, including :
- locating open reading frames
- translating the DNA sequence into amino acid sequence

1. Launch the APE program. Select the Open file icon.

2. Find and load the sequence file B.cer Amy.str. This is the DNA sequence for the gene that
codes for an enzyme call -amylase in Bacillus cereus.

3. Note down : (a) The length of the sequence = _______________

(b) The start codon =

(c) The stop codon =

4. Find out how many restriction sites for each of these enzymes in the sequence :

EcoRI DraI BamHI

ApoI HindIII RsaI

5. If we digest this DNA using PsiI,

(a) How many fragments will be produced ?

(b) What are the sizes of these fragments ?

Locating ORFs

6. Close all other windows except the editor window (the one showing your sequence)

7. Select the ORFs fucntion from the menu bar on top. Select ORF Map.

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8. You should see a new window showing the six possible open reading

9. What is a reading frame ?

10. Why are there altogether six reading frames?

11. In each reading frame, a short vertical line represents the presence of a ________________

while a long vertical line represents a ___________________

12. Which of the six do you think is the correct reading frame ? Why ?

13. Close all other windows. Select ORFs again from the top bar menu. Now select Translate.

Select all the options as shown below

14. A new window will open up :

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15. This is the translation of the DNA sequence into its corresponding amino acid sequence.
Note that each codon has is made up of three consecutive nucleotides.

16.
How many codons are there ? How many amino acids ?

What are the amino acids at these positions ?

(a) 10 = (b) 85 = (c) 218 = (d) 431 =

Exercise : Virtual mutation

17. We will change (‘mutate’) part of the DNA sequence and see how it affect the protein. Do
steps no. 22 to 27 below. Note down what happen to the protein and state what type of
mutation it represent.

18. In the editor window, select nucleotide no 36. Change it to an A. Then translate the new
sequence.

What type of mutation have you created? What are the consequences of this mutation?

19. Select Edit and Undo Typing to restore the original sequence (or simply press Ctrl-Z).
Now change nucleotide no 68 to an A.

What type of mutation have you created? What are the consequences of this mutation?
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20. Restore the sequence (Ctrl-Z). Change nucleotide no 13 to an A.

21. Restore the sequence. Delete the G at position no 61.

22. Restore the sequence. Add a C add position no 60.

23. Restore the sequence. Select nucleotides no 121 to 140 and delete the entire selection.
Then make a few random changes. Save this sequence under your name.

Sequence alignment

24. We will now learn how align two sequences. This feature is useful when comparing cloasely
related genes.

25. Open a new editor window. Load the B cer Amy sequence again.

26. Click once on each of the editor window of both sequences. Select the Align Sequences icon.

27. The Align Sequences dialogue box will open up. Make sure both sequences are selected for
alignment and click OK.

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28. A new window will open, displaying the alignment of both sequences to each other.

The Sequence Alignment function compares both sequences and try to match similar
stretches of nucleotides. The red areas indicates sequences that could not be matched
(mismatches).

Example

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