MASENO UNIVERSITY

SCHOOL OF PUBLIC HEALTH AND COMMUNITY DEVELOPMENT

DEPARTMENT OF BIOMEDICAL SCIENCE AND TECHNOLOGY

PMT 324: MOLECULAR BIOLOGY OF THE GENE

PRACTICAL 1: PURIFICATION OF TOTAL DNA FROM WHOLE BLOOD

PRINCIPLE
Depending on the starting material, samples are digested with Proteinase K in either the
supplied Digestion or Lysis Solution. RNA is removed by treating the samples with RNase A.
The lysate is then mixed with ethanol and loaded on the purification column where the DNA
binds to the silica membrane. Impurities are effectively removed by washing the column with
the prepared wash buffers. Genomic DNA is then eluted under low ionic strength conditions
with the Elution Buffer.

Materials and reagents

- Whole blood
- Normal saline
- BioFluid and Cell Buffer
- Proteinase K
- Genomic binding buffer
- DNA pre-Wash buffer
- DNA Wash buffer
- Elution Buffer
- Collection tubes
- Zymo-spin Column
- Microcentrifuge tubes
- Centrifuge
- Vortexer
- Micropipettes
- Pipet tips
- Water bath/heating block
- Stop watch

Procedure
1. Pippette 200 µl of blood sample to a microcentrifuge tube.
2. Add 200 µl of BioFluid and Cell Buffer.
3. Add 20 µl of proteinase K .
4. Vortex for 10 - 15 seconds and then incubate the tube for 10 minutes at 55°C.
5. Add 200µl of Genomic binding buffer to 200µl of the digested sample. Mix by vortexing for 10
seconds to obtain a uniform suspension.
6. Transfer the mixture to a Zymo-spin Column in a collection tube. Centrifuge the column for 1
minute at 12,000g. Discard the collection tube containing the flow-through solution.
7. Place the Zymo-spin Column into a new collection tube, add 400µl DNA Pre-Wash buffer,
centrifuge for 1 minute at 12,000g. Empty the flow-through in the collection tube. Do not discard
the collection tube.
8. Add 700µl g-DNA Wash buffer and centrifuge for 1 minute at 12,000g. Empty the flow-through
in the collection tube. Do not discard the collection tube.
9. Add 200µl g-DNA Wash buffer and centrifuge for 1 minute at 12,000g. Discard the collection
tube containing the flow-through solution.
10. Transfer the Zymo-spin Column into a clean microcentrifuge tube and add 50µl of DNA Elution
buffer to the center of the column membrane to elute genomic DNA.
11. Incubate at room temperature for 5 minute and then centrifuge for 1 minute at 14,000g to elute
the DNA.
12. Discard the purification column. Use the purified DNA immediately for molecular based
applications or store at -20 °C for future use.
 MASENO UNIVERSITY
SCHOOL OF PUBLIC HEALTH AND COMMUNITY DEVELOPMENT
DEPARTMENT OF BIOMEDICAL SCIENCE AND TECHNOLOGY

PMT 324: MOLECULAR BIOLOGY OF THE GENE

PRACTICAL 2: PCR

Overview
PCR is an in vitro technique that imitates nature’s ability to replicate DNA. The technology generates
multiple copies of specific nucleotide sequence from a target organism. It also provides a mechanism to
detect extremely low concentrations of target organisms with high specificity.

Materials and reagents

- Micropipettes
- Pipette tips
- Centrifuge
- Amplification tubes
- DNA extract
- dNTPs
- 10X buffer
- MgCl2
- Forward Primer
- Reverse Primer
- PCR water
- Taq polymerase
- Tubes
- Biohazard container

Procedure
1. Pipette 4 µl of dNTPs into a reaction tube
2. Add 2.5µl of 10X buffer to the tube
3. Add 1.5µl of MgCl2
4. Add 1.5 µl of primer 1 (forward) and primer 2 (reverse) respectively to the mixture
5. Pipette 14.1 µl of PCR water and add to the mixture
6. To the mixture, add 0.4µl of Taq polymerase (enzyme)
7. Vortex the master mix and short spin in a mini centrifuge
8. Pipette 23µl of the master mix to the amplification tubes
9. Add 2 µl of the DNA extract to the amplification tubes and load onto the thermal cycler

PCR Conditions
1. 94° C for 2 minutes
2. 40 cycles of:
- 94° C for 45 seconds
- 62° C for 45 seconds
- 72° C for 2 minutes
3. 72° C for 7 minutes
4. 4° C forever
 MASENO UNIVERSITY
SCHOOL OF PUBLIC HEALTH AND COMMUNITY DEVELOPMENT
DEPARTMENT OF BIOMEDICAL SCIENCE AND TECHNOLOGY

PMT 324: MOLECULAR BIOLOGY OF THE GENE

PRACTICAL 3: GEL ELECTROPHORESIS OF DNA SAMPLES

Introduction
Nucleic acid samples are often subjected to gel electrophoresis to characterize the size and number of
different fragments in the sample. In this exercise, you will be using DNA samples generated from PCR.
Some of which have been digested with restriction enzymes (bacterial enzymes capable of cutting the
DNA molecule at specific sites). These samples are then mixed with a tracking dye so you can keep track
of their migration since you can't really see the DNA molecules until they have been further processed.
The samples are subjected to agarose gel electrophoresis (electrical current is passed through a buffer
system and gel).

Principle
When charged molecules are placed in an electrical field, the molecules will migrate towards one of the
electrodes, depending on the net charge of the molecule. Nucleic acids are negatively charged so they
will migrate toward the positive electrode.

Separation of nucleic acid molecules of different size or conformation is achieved by forcing the
molecules to migrated through a support matrix (in this case: agarose gel) in the electrical field. This
matrix acts like a sieve to allow small molecules to migrate faster than larger molecules. The result is that
molecules separate in the matrix according to their relative size and shape.

This exercise will use gel electrophoresis to examine the fragments present in several DNA samples with
and without enzyme digestion. In this way you will gain experience in pouring gels, loading DNA
samples, and visualizing DNA bands on the gels. Due to the very small volumes of samples to be applied
to the gel, samples are routinely handled with a manual micropipette that uses a disposable plastic tip to
handle a 10 µl to 20 µl sample.

Objectives:
 Explain how molecules migrate through agarose gels based on their charge and molecular weight
 Prepare a solution in which the concentration is given as a percentage
 Pour, load, and run an agarose gel

Requirements
- One gel electrophoresis box with electrode cords
- One gel former with comb (to form wells in gel)
- Power supply box
- Flask of agarose in the buffer (1x)
- DNA samples
- Micropipettes
- Pipette tips
Procedure:

1. Heat the flask of buffer and agarose in the microwave until it starts to boil. Take it out and swirl
gently. Make sure you use the heat protecting glove at the microwave station because the flask
will be HOT. Repeat this procedure until all the agarose is melted and there are no little specks
in the melted agar.

2. Cool the flask of agar until it is comfortable to touch (55 oC). Add 2 µl of Ethidium bromide to
the agarose and mix thoroughly. This is the stain to detect the DNA. Ethidium bromide is a
mutagen (comparable to a pack of cigarettes) and solutions containing Ethidium bromide should
not be handled with bare hands.

3. Set the comb in the groove at the black end of your gel tray. Make a border using masking tape
on the open ends of the gel try. Pour the melted agar into the tray until it comes up about a little
less than 1/3 the length of a comb tooth (approximately 3 mm). Allow the gel to cool (it will turn
opaque when solidified).

4. When cool, take a small amount of buffer and pour it on the gel as a lubricant. Remove the comb
straight up and you will be left with tiny wells where the teeth were. Remove the masking tape.
Do not tip the gel tray because the gel may "slip" off and be ruined.

5. Place the gel tray between the two posts in the electrophoresis box with the black tape side on the
same side as the black dot on the back of the box. Your wells on the gel should also be on this
side (negative side).

6. Pour buffer in the box until it just barely covers the gel.

7. Load 10 µl of each DNA sample in each well in the gel.

8. Close the box by lining up the electrode posts in the back of the lid with the back of the box and
sliding the cover toward you as you hold the box. Make sure the black lead (wire) is plugged in
the negative hole and the red lead is plugged into the positive receptacle on the power supply box.

9. Turn on the power supply switch on the power box and adjust the voltage to 100-120 volts. The
channel switch lets you read the voltage for either gel box depending on the location of the
switch. Make sure the position of the switch matches your gel box when adjusting the voltage.
Since each power box can accommodate two gel boxes, two groups will use the same power
supply box.

10. Each DNA sample has already been mixed with a blue loading dye which allows you to watch the
migration through the gel. When the furthest band of blue is about 3/4 of the way to the end of
the gel, turn off the power supply and you are ready to view the sample under the UV
transilluminator (about 45 minutes at 120 volts). A photograph can now be taken for a permanent
record.

Prepared by:
1. Imelda Mboya Signature……….…………………. Date………………..

2. Jackline Omundi Signature……….…………………. Date……………….

Approved by:
Ag. Chief Technologist, BMST
Signature……………..………………….Date………………..